Chimeric factor h binding proteins (fhbp) and methods of use

ABSTRACT

Chimeric fHbps that can elicit antibodies that are bactericidal for different fHbp variant strains of  N. meningitidis , and methods of use, are provided.

CROSS REFERENCED TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/174,424 filed Apr. 30, 2009, which is incorporated herein byreference in its entirety and for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Public HealthService grant nos. R01AI46464 and C06 RR16226. The government hascertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to vaccines for diseases caused by Neisseriameningitidis.

INTRODUCTION

Neisseria meningitidis is a Gram-negative bacterium which colonizes thehuman upper respiratory tract and is responsible for worldwide sporadicand cyclical epidemic outbreaks of, most notably, meningitis and sepsis.The attack and morbidity rates are highest in children under 2 years ofage. Like other Gram-negative bacteria, Neisseria meningitidis typicallypossess a cytoplasmic membrane, a peptidoglycan layer, an outer membranewhich together with the capsular polysaccharide constitute the bacterialwall, and pili, which project into the outside environment. Encapsulatedstrains of Neisseria meningitidis are a major cause of bacterialmeningitis and septicemia in children and young adults. The prevalenceand economic importance of invasive Neisseria meningitidis infectionshave driven the search for effective vaccines that can confer immunityacross different strains, and particularly across genetically diversegroup B strains with different serotypes or serosubtypes.

Factor H Binding Protein (fHbp, also referred to in the art aslipoprotein 2086 (Fletcher et al, Infect Immun 2004; 72:2088-2100),Genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. J Exp Med2003; 197:789-99) or “741”) is an N. meningitidis protein which isexpressed in the bacterium as a surface-exposed lipoprotein. Animportant function of fHbp is to bind human complement 1H, whichdown-regulates complement activation. Binding of fH to the bacterialsurface is an important mechanism by which the pathogen survives innon-immune human serum or blood and evades innate host defenses.

From analysis of 71 N. meningitidis strains in the first study, and morethan 200 strains in the second study, representative of its genetic andgeographic diversity, N. meningitidis strains have been sub-divided intothree fHbp variant groups (referred to as variant 1 (v.1), variant 2(v.2), and variant 3 (v.3)) based on amino acid sequence variability andimmunologic cross-reactivity (Masignani et al. J Exp Med 2003;197:789-99). Other workers (Fletcher et al, 2004) have subdivided theprotein into two sub-families designated A (which includes v.2 and v.3of Masignani) and B (v.1). Variant 1 strains account for about 60% ofdisease-producing group B isolates (Masignani et al. 2003, supra).Within each variant group, there is on the order of about 92% or greaterconservation of amino acid sequence. Specifically, conservation withineach variant group ranges between 89 and 100%, while between the variantgroups (e.g., between v.1 and v.2) the conservation can be as low as59%. The protein is expressed by all known strains of N. meningitidis.

There remains a need for a single fHbp polypeptide that can elicitbactericidal antibody responses that are effective against a broadspectrum of strains expressing different fHbp variants.

SUMMARY

Chimeric fHbps that can elicit antibodies that are bactericidal fordifferent fHbp variant strains of N. meningitidis, and methods of use,are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G is Table 7 that lists the source strains and characteristicsof about 69 unique factor H binding proteins.

FIG. 2A-2E is Table 8 that lists unique sequences in fHbp variablesegment A through E and number of unique peptides from each variantgroup containing the segment ID. Segment ID is an identifier for eachvariable segment. Distinct sequence variants were assigned a uniqueidentifier beginning with a letter, A through E, to represent thesegment; followed by an α or β to indicate homology of the segment witheither the corresponding fHbp segment from peptide ID 1 (fHbp v.1) orpeptide ID 28 (fHbp v.3), respectively, followed by a number for eachdistinct sequence. Numbers in variant groups 1, 2, and 3 are providedfor some segment IDs of each segment (A through E) in subtables.

FIG. 3 is a phylogram of fHbp based on 69 unique amino acid sequences.

FIG. 4, panel A is a schematic representation of fHbp showing positionsof linkers, which are also referred herein as invariant segment (I),shown as vertical rectangles, together with variable segments (V). PanelB is a table listing the amino acid sequence for each invariant segment.

FIG. 5 is a phylogram of unique fHbp amino acid sequences in variablesegments A (residues 8-73) (panel A) and B (residues 79-93) (panel B).

FIG. 6 is a phylogram of unique fHbp amino acid sequences in variablesegments C (residues 98-159) (panel A) and D (residues 162-180) (panelB).

FIG. 7 is a phylogram of unique fHbp amino acid sequences in variablesegment E (residues 186-253).

FIG. 8, panel A is a schematic representation of 9 fHbp modulararchitectures deduced from phylogenic analysis, including the morecommon 6 modular group types. Gray segments represent α progenitorsequences homologous to peptide ID 1 while white segments represent βprogenitor sequences homologous to peptide 28. About 94% of the uniquefHbp amino acid sequences analyzed herein belong to one of the first sixmodular group types (I, II, III, IV, V, and VI). Panel B, Four of theanalyzed fHbps are classified as one of three modular architecturesshown. The architectures contain junction points within a variablesegment, designated by arrows. Panel C is a table listing the amino acidresidues at which an α progenitor sequence switches to a β or vice versa(J₁, J₂, and J₃) within a variable segment.

FIG. 9 presents structural models of factor H binding protein based onthe coordinates of fHbp in a complex with a fragment of human factor H(Schneider et al. (2009) Nature 458:890-3). Left models in each of thefollowing panels show fHbps facing membrane side; center models arefacing exposed side; and the right models depict membrane side of thefHbp down. Panel A, Cartoon representation depicting the two structuraldomains. B, Space-filling model with factor H-binding residues depictedin black and the amino acid residues of the invariant segments in white.C, Space-filling model with the amino acid residues of the invariantsegments depicted in white as in panel B, and the residues affecting theepitopes of anti-fHbp mAbs shown in black.

FIG. 10A-10I is Table 9 that lists certain characteristics of uniquefactor H binding protein variants use in an analysis that include 275peptide IDs. Sequence identifiers for N-terminal variable sequences areas follows: GGGS (SEQ ID NO: 7), SGSGG (SEQ ID NO: 8), GGGSGG (SEQ IDNO:9), GGGSGS (SEQ ID NO:10), GSGG (SEQ ID NO:11), GGGSGGGG (SEQ IDNO:12), GGGSGGGSGG (SEQ ID NO:13), and GGSGG (SEQ ID NO:14).

FIG. 11 shows fHbp expression measured by immunoblotting with infrareddetection. Panels A and C, Recombinant proteins in modular groups I(ID 1) or VI (ID 77), or heat-killed bacterial cells from strainsexpressing fHbp in the corresponding modular groups. The modular group Iproteins were detected with murine anti-fHbp mAb JAR 5, which recognizesnearly all fHbps in modular groups I and IV. The modular group VIproteins were detected with anti-fHbp mAb JAR 31, which recognizesnearly all proteins in modular groups II, III, V and VI (See Table 6 inexample section). Panels B and D, Standard curves from the correspondingbinding of the recombinant proteins shown in Panels A and C.

FIG. 12 shows the frequencies of fHbp modular groups amongsystematically collected N. meningitidis group B case isolates. Data arefrom sequences of isolates collected in the United States (N=432),United Kingdom (N=536) and France (N=244) reported by Murphy et al [16],and newly obtained sequences of 143 additional U.S. isolates fromCalifornia (2003-2004), Maryland (1995 and 2005), and pediatrichospitals in 9 states (2001-2005).

FIG. 13 shows serum bactericidal activities of serum pools from miceimmunized with fHbps from modular groups Ito VI. The black barsrepresent the median titers of 3 to 4 serum pools for each modular grouptested against homologous test strains. The white bars represent themedian titer of the respective heterologous serum pools against the teststrain. +, refers to relative expression of fHbp by each of the strains;strains with +/− representing low fHbp-expressing strains (see values inTable 5 in example section)

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantigen” includes a plurality of such antigens and reference to “theprotein” includes reference to one or more proteins, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The present disclosure provides chimeric fHbps that can elicitantibodies that are bactericidal for different fHBP variant strains ofN. meningitidis, and methods of use.

DEFINITIONS

“Factor H Binding Protein” (fHbp), which is also known in the literatureas GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and “741”refers to a polypeptide of N. meningitidis that is a lipoproteinpresented on the surface of the bacterium. N. meningitidis strains havebeen sub-divided into three fHbp variant groups (referred to as variant1 (v.1), variant 2 (v.2), and variant 3 (v.3) in some reports (Masignaniet al. 2003, supra) and Family A and B in other reports (see, e.g.,Fletcher et al. 2004 Infect Immun 2088-2100)) based on amino acidsequence variability and immunologic cross-reactivity (Masignani et al.J Exp Med 2003; 197:789-99). Each unique fHbp found in N. meningitidisis also assigned a peptide ID according to neisseria.org, as shown inFIGS. 1A-1G (Table 7) and FIGS. 10A-10I (Table 9). For clarity, thepresent disclosure uses the v.1, v.2 and v.3 terminology. Because thelength of variant 2 (v.2) fHbp protein (from strain 8047, peptide ID 77)and variant 3 (v.3) fHBP (from strain M1239, peptide ID 28) differ by −1and +7 amino acid residues, respectively, from that of MC58 (peptide ID1), the numbering used to refer to residues for v.2 and v.3 fHbpproteins differs from numbering based on the actual amino acid sequencesof these proteins. Thus, for example, reference to a leucine residue (L)at position 166 of the v.2 or v.3 fHBP sequence refers to the residue atposition 165 of the v.2 protein and at position 173 in the v.3 protein.

The term “heterologous” or “chimeric” refers to two components that aredefined by structures derived from different sources. For example, where“heterologous” is used in the context of a chimeric polypeptide, thechimeric polypeptide includes operably linked amino acid sequences thatcan be derived from different polypeptides of different phylogenicgroupings (e.g., a first component from an α and a second component froma β progenitor amino acid sequences). Similarly, “heterologous” in thecontext of a polynucleotide encoding a chimeric polypeptide includesoperably linked nucleic acid sequence that can be derived from differentgenes (e.g., a first component from a nucleic acid encoding a fHbp v.1polypeptide and a second component from a nucleic acid encoding a fHbpv.2 polypeptide). Such chimeric polypeptides as described herein providefor presentation of epitopes in a single polypeptide that are normallyfound in different polypeptides. Other exemplary “heterologous” nucleicacids include expression constructs in which a nucleic acid comprising acoding sequence is operably linked to a regulatory element (e.g., apromoter) that is from a genetic origin different from that of thecoding sequence (e.g., to provide for expression in a host cell ofinterest, which may be of different genetic origin relative to thepromoter, the coding sequence or both). For example, a T7 promoteroperably linked to a polynucleotide encoding an fHbp polypeptide ordomain thereof is said to be a heterologous nucleic acid. “Heterologous”in the context of recombinant cells can refer to the presence of anucleic acid (or gene product, such as a polypeptide) that is of adifferent genetic origin than the host cell in which it is present. Forexample, a Neisserial amino acid or nucleic acid sequence of one strainis heterologous to a Neisserial host of another strain.

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” av.1 fHbp) is meant to indicate that the polypeptide or nucleic acid hasa sequence that is based on that of a reference polypeptide or nucleicacid (e.g., a naturally occurring fHbp protein or encoding nucleicacid), and is not meant to be limiting as to the source or method inwhich the protein or nucleic acid is made. “Derived from” in the contextof bacterial strains is meant to indicate that α strain was obtainedthrough passage in vivo, or in in vitro culture, of a parental strainand/or is a recombinant cell obtained by modification of a parentalstrain.

“Conservative amino acid substitution” refers to a substitution of oneamino acid residue for another sharing chemical and physical propertiesof the amino acid side chain (e.g., charge, size,hydrophobicity/hydrophilicity). “Conservative substitutions” areintended to include substitution within the following groups of aminoacid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr. Conservative amino acid substitutions in thecontext of a chimeric fHBP disclosed herein are selected so as topreserve presentation of an epitope of interest. Guidance for suchsubstitutions can be drawn from alignments of amino acid sequences ofpolypeptides presenting the epitope of interest.

The term “protective immunity” means that a vaccine or immunizationschedule that is administered to a mammal induces an immune responsethat prevents, retards the development of, or reduces the severity of adisease that is caused by Neisseria meningitidis, or diminishes oraltogether eliminates the symptoms of the disease. Protective immunitycan be accompanied by production of bactericidal antibodies. It shouldbe noted that production of bactericidal antibodies against Neisseriameningitidis is accepted in the field as predictive of a vaccine'sprotective effect in humans. (Goldschneider et al., 1969, J. Exp. Med.129:1307; Borrow et al. 2001 Infect Immun. 69:1568).

The phrase “a disease caused by α strain of capsular group B ofNeisseria meningitidis” encompasses any clinical symptom or combinationof clinical symptoms that are present in an infection of a human with amember of capsular group B of Neisseria meningitidis. These symptomsinclude but are not limited to: colonization of the upper respiratorytract (e.g. mucosa of the nasopharynx and tonsils) by a pathogenicstrain of capsular group B of Neisseria meningitidis, penetration of thebacteria into the mucosa and the submucosal vascular bed, septicemia,septic shock, inflammation, haemmorrhagic skin lesions, activation offibrinolysis and of blood coagulation, organ dysfunction such as kidney,lung, and cardiac failure, adrenal hemorrhaging and muscular infarction,capillary leakage, edema, peripheral limb ischaemia, respiratorydistress syndrome, pericarditis and meningitis.

The phrase “broad spectrum protective immunity” means that a vaccine orimmunization schedule elicits “protective immunity” against at leastmore than one strain (and can be against at least two, at least three,at least four, at least five, against at least eight, or more strains)of Neisseria meningitidis, wherein each of the strains expresses adifferent fHbp subvariant or fHbp variant. The present disclosurespecifically contemplates and encompasses a vaccine or vaccinationregimen that confers protection against a disease caused by a member ofany capsular group (e.g., A, B, or C), with protection against diseasecaused by a capsular group B strain of Neisseria meningitidis being ofinterest due to the epidemiological prevalence of strains causingdisease with this capsular group and lack of broadly effective group Bvaccines.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, in the context of an antigen (e.g., a polypeptideantigen) refers to a binding reaction which is based on and/or isprobative of the presence of the antigen in a sample which may alsoinclude a heterogeneous population of other molecules. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular antigen or antigens in a sample and do not bind in asignificant amount to other molecules present in the sample.“Specifically binds to an antibody” or “specifically immunoreactivewith” in the context of an epitope of an antigen (e.g., an epitope of apolypeptide) refers to a binding reaction which is based on and/or isprobative of the presence of the epitope in an antigen (e.g.,polypeptide) which may also include a heterogeneous population of otherepitopes, as well as a heterogeneous population of antigens. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular epitope of an antigen and do not bind in a significant amountto other epitopes present in the antigen and/or in the sample.

The phrase “in a sufficient amount to elicit an immune response” meansthat there is a detectable difference between an immune responseindicator measured before and after administration of a particularantigen preparation Immune response indicators include but are notlimited to: antibody titer or specificity, as detected by an assay suchas enzyme-linked immunoassay (ELISA), bactericidal assay, flowcytometry, immunoprecipitation, Ouchter-Lowny immunodiffusion; bindingdetection assays of, for example, spot, Western blot or antigen arrays;cytotoxicity assays, etc.

A “surface antigen” is an antigen that is present in a surface structureof Neisseria meningitidis (e.g. the outer membrane, capsule, pili,etc.).

“Isolated” refers to an entity of interest that is in an environmentdifferent from that in which the compound may naturally occur.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.”

“Enriched” means that a sample is non-naturally manipulated (e.g., by anexperimentalist or a clinician) so that a compound of interest ispresent in a greater concentration (e.g., at least a three-fold greater,at least 4-fold greater, at least 8-fold greater, at least 64-foldgreater, or more) than the concentration of the compound in the startingsample, such as a biological sample (e.g., a sample in which thecompound naturally occurs or in which it is present afteradministration), or in which the compound was made (e.g., as in abacterial polypeptide, antibody, chimeric polypeptide, and the like)

A “knock-out” or “knockout” of a target gene refers to an alteration inthe sequence of the gene that results in a decrease of function of thetarget gene, e.g., such that target gene expression is undetectable orinsignificant, and/or the gene product is not functional or notsignificantly functional. For example, a “knockout” of a gene involvedin LPS synthesis indicates means that function of the gene has beensubstantially decreased so that the expression of the gene is notdetectable or only present at insignificant levels and/or a biologicalactivity of the gene product (e.g., an enzymatic activity) issignificantly reduced relative to prior to the modification or is notdetectable. “Knock-outs” encompass conditional knock-outs, wherealteration of the target gene can occur upon, for example, exposure to apredefined set of conditions (e.g., temperature, osmolarity, exposure tosubstance that promotes target gene alteration, and the like. A“knock-in” or “knockin” of a target gene refers to a genetic alterationin a host cell genome that that results in an increase in a functionprovided by the target gene.

“Non-naturally occurring”, as used herein, refers to a protein (e.g.fHbp) that is not normally found in nature and is instead artificiallyproduced and/or modified by a human. A non-naturally occurring subjectfHbp can be made via chemical synthesis or recombinant methods. Forexample, “non-naturally occurring chimeras” refers to “man-madechimeras” and encompass fHbp with heterologous components that are notfound in nature.

fHbp and fHbp-Encoding Nucleic Acids

Before describing further exemplary chimeric fHbps contemplated by thepresent disclosure, it is helpful to describe naturally-occurring fHbps.

For convenience and clarity, the native amino acid sequence of the v.1fHBP of the N. meningitidis strain MC58 (peptide ID 1) was arbitrarilyselected as a reference sequence for all native v.1, v.2, and v.3 fHbpamino acid sequences, as well as for the chimeric fHbps describedherein. Unless otherwise noted, amino acid residue positions are alsoreferred herein with reference to peptide ID 1. The sequence of peptideID 1 is presented below:

(SEQ ID NO: 1) CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

Despite the diversity of variant groups and other classification schemesfor the sequences listed in FIGS. 1A-1G (Table 7) and FIGS. 10A-10I(Table 9), a first analysis of 69 unique fHbp sequences and a secondanalysis of all 275 peptide IDs, as described in the Examples belowrevealed that several different stretches of amino acid sequences withinthe fHbps are conserved among all the fHbps analyzed. As shown by thephylogenic studies presented in the Examples section below, theseconserved amino acid sequences separate fHbp into modular segments ofamino acid sequences that cluster in the phylogram to define twoprogenitor sequences from which each variable segment may be derived. Asillustrated in FIGS. 5-7, each of the variable segments in fHbps can beclassified as being of one of the two progenitor amino acid sequences:designated for convenience, α or β.

The nomenclature architecture used to characterize fHbp based on modularsegments is illustrated in FIG. 4, panel A. The rectangles in FIG. 4,panel A represent the conserved sequences (I) and the variable segmentsare designated as the N-terminal element, V_(A), V_(B), V_(C), V_(D), orV_(E) segments. The amino acid sequence of the conserved sequences arelisted in FIG. 4, panel B.

As such, naturally occurring fHbps can be defined by the formula:I₁-Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-h, wherein “Nte”refers to the N-terminal element, “I” refers to an “invariant” segment,and “V” refers to a variable segment of either an α or a β progenitortype.

As disclosed below in more detail, the non-naturally occurring chimericfHbps of the present disclosure can be constructed by variouscombinations of the V segments which are flanked by I segments. Theinvariant and variable segments which can be used in construction ofsuch chimeric fHbps are described from N-terminus to C-terminus in moredetail below. This modular classification can also be found on theNeisseria.org website(neisseria.org/perl/agdbnet/agdbnet.pl?file=nm_fhbp.xml). On the websitethe progenitor groups α and β as described below are referred to 1 and2, respectively. For example, V_(A)α2-V_(B)α1-V_(C)β5-V_(D)α5-V_(E)α8becomes A1.2-B1.1-C2.5-D1.5-E1.8.

Invariant Segments

fHbps contain amino acid sequences that are conserved among all fHbpamino acid sequences analyzed herein. The conserved sequences flankvariable segments that are derived from either the α or β progenitoramino acid sequences. These conserved sequences are referred to hereinas “invariant sequences” or “invariant segments”, and may serve as sitesof recombination in naturally-occurring fHbps. Where these invariantsegments are flanked by variable segments, the invariant segments may bereferred to as “linker” sequences. While these sequences exhibit aminoacid sequence identity between the fHbps disclosed herein, such aminoacid sequences may still tolerate certain conservative amino acidsubstitutions. As such, the term “invariant segment” as used hereinshould not be construed to be limited to the specific amino acidsequences described herein, and may at least contain one or moreconservative amino acid substitutions.

As illustrated in FIG. 4, the most N-terminal invariant segment found innaturally-occurring fHbps starts at residue 1 and is of the amino acidsequence CSSG (SEQ ID NO:2) Immediately C-terminal to the firstinvariant segment CSSG (I₁) is a variable N-terminal element of about 1to about 6 glycine or serine residues. The various N-terminal elementsare listed in FIG. 1A-1G (Table 7) and FIGS. 10A-10I (Table 9). Thesecond invariant segment (I₂) is of the amino acid sequence GG, which ispositioned C-terminal to the N-terminal element and starts at residue 6,7, or 11, depending on the length of the N-terminal element (e.g., inpeptide ID 28 of variant 3, I₂ starts at residue 11). The next invariantsegment, I₃, is defined by the amino acid sequence of SRFDF (SEQ IDNO:3) and is positioned at residue 74, C-terminal to I₂ and a variablesegment. I₄, which is C-terminal to I₃, is defined by the amino acidsequence GEFQ (SEQ ID NO:4) and is positioned at residue 94. I₅ isdefined by the amino acid sequence DD and is C-terminal to I₄ at residue159. I₆, positioned C-terminal to I₅, is defined by IEHLK (SEQ ID NO:5)or IEHLE (SEQ ID NO: 6) and starts at residue 180. Lastly, I₇, the mostC-terminal invariant segment in naturally-occurring fHbps, starts atresidue 252 and is defined by the amino acid sequence of KQ.

The residue position listed above at which each invariant segment startsmay be shifted from 1 to 8 residues depending on the length of theN-terminal element and the amino acid sequence of the variable segments.As noted above, for convenience and clarity residue numbering used inreference to non-naturally occurring chimeric fHbps throughout thepresent disclosure is based on the amino acid sequence numbering of thefHbp of MC58 (peptide ID 1), which is a variant 1 strain.

Based on the sequences analyzed herein, the length of the invariantsegments ranges from about 2 amino acid residues to about 5 amino acidresidues.

Variable Segments

As noted above, variable segments (V) are flanked by linkers, alsoreferred herein as invariant segments (I). In addition to a variableN-terminal element, there are five variable segments in an fHbp and theyare designated from N-terminus to C-terminus as V_(A), V_(B), V_(C),V_(D), and V_(E), each flanked by the invariant segments discussedabove. As mentioned previously, each of the variable segments may beclassified as being of one of the two progenitor amino acid sequences: αor β. For example, V_(A)α indicates that variable segment A is derivedfrom an α progenitor amino acid sequence while V_(A)β refers to avariable segment A derived from a β progenitor amino acid sequence. In amore specific example, V_(A)α1 denotes that the variable segment A is ofthe amino acid sequence set forth as Segment ID A.α.1 in FIG. 2A (Table8, segment A), which is one allele of the α progenitor sequences ofvariable segment A. As described below, the chimeric fHbp of the presentdisclosure contain a non-naturally occurring combination of variablesegments in which each segment may be independently selected from eitheran α and a β progenitor type and/or a non-naturally occurringcombination of any alleles of the corresponding segment shown in FIGS.2A-2E, Table 8.

The α and β progenitor variable segments are each defined by certainsignature amino acid residues. In addition, the α progenitor variablesegments exhibit sequence similarity to fHbp of variant 1 group (e.g.peptide ID 1), while the β progenitor variable segments exhibit sequencesimilarity to fHbp of variant 3 group (e.g. peptide ID 28). Signatureamino acid residues of α or β progenitors for each variable segment arebe discussed below.

In addition to being defined by their corresponding amino acid signaturesequence, variable segments of the same progenitor type share amino acidsequence identity of at least 80%, at least 85%, at least 90%, at least95%, and can be 100%. When amino acid sequences of the same variablesegments of different progenitor types are compared, the amino acidsequence identity drops by at least 10% to 40%.

See Table 4 in Example section below for amino acid identities withinand between sequence groups by segment.

Various alleles for each variable segment and their sequences arepresented in FIG. 2A-2E (Table 8) and are described in detail below.

The most N-terminal variable segment is referred to herein as theN-terminal element (Nte) (FIG. 4). The various N-terminal elements foundin the fHbps analyzed are listed in FIGS. 1A-1G (Table 7) and also inFIGS. 10A-10I (Table 9). They range in length from 1 to 6 residues ofglycine or a combination of glycine and serine.

The variable segment V_(A) starts at residue 7, C-terminal to I₂, andends at residue 73, N-terminal to I₃. V_(A) of an α progenitor (V_(A)α)comprises an amino acid sequence that is about 89 to 100% identical toSegment ID A.α.1 as presented in FIG. 2A (Table 8): VAADIGAGLADALTAPLDHK DKSLQSLTLD QSVRKNEKLK LAAQGAEKTY GNGDSLN TGKLKNDKV (SEQ IDNO:15). V_(A)α is further characterized by signature amino acid residuesQSV, bolded in sequence. Certain residues that may have alternativeamino acid residues among V_(A)α of different fHbps are underlined.

V_(A) of a β progenitor (V_(A)β) comprises an amino acid sequence thatis about 89 to 100% identical to Segment ID A.β.1 as set forth in FIG.2A (Table 8): VAADIGTGLA DALTAPLDHK DKGLKSLTLE DSIPQNGTLT LSAQGAEKTFKAGDKDNSLN TGKLKNDKI (SEQ ID NO:67). V_(A)β is further characterized bysignature amino acid residues DSI and/or KDN, bolded in sequence.Certain residues that have alternative or deleted amino acid residuesamong V_(A)β of different fHbps are underlined.

The variable segment V_(B) starts at residue 79, and ends N-terminal towhere I₄ is located, at residue 93. V_(B) of an α progenitor (V_(B)α)comprises an amino acid sequence that is about 80% to 100% identical toSegment ID B.α.1 as set forth in FIG. 2B (Table 8): IRQIEVDGQL ITLES(SEQ ID NO:85). V_(B)α is further characterized by signature amino acidresidues IRQ, bolded in the sequence. Certain residues that havealternative or deleted amino acid residues among V_(B)α of differentfHbps are underlined.

V_(B) of a β progenitor (V_(B)β) comprises an amino acid sequence thatis about 100% identical to Segment ID B.β.1 as set forth in FIG. 2B(Table 8): VQKIEVDGQT ITLAS (SEQ ID NO:95). V_(B)β is furthercharacterized by signature amino acid residues VQK, bolded in thesequence.

The variable segment V_(C) starts around residue 98, C-terminal to I₄and ends at residue 158, N-terminal to I₅. V_(C) of an α progenitor(V_(C)α) comprises an amino acid sequence that is about 85 to 100%identical to Segment ID C.α.1 as presented in FIG. 2C (Table 8):VYKQSHSALT ALQTEQVQDS EHSGKMVAKR QFRIGDIAGE HTSFDKLPEG GRATYRGTAF GS(SEQ ID NO:96). V_(C)α is further characterized by signature amino acidresidues QDS, bolded in the sequence. Certain residues that havealternative or deleted amino acid residues among V_(C)α of differentfHbps are underlined.

V_(C) of a β progenitor (V_(C)β) comprises an amino acid sequence thatis about 93 to 100% identical to Segment ID C.β.1 as presented in FIG.2C (Table 8): IYKQDHSAVV ALQIEKINNP DKIDSLINQR SFLVSGLGGE HTAFNQLPGGKAEYHGKAF SS (SEQ ID NO:152). V_(C)β is further characterized bysignature amino acid residues NNP, bolded in sequence. This signature isfound in all of V_(C)β amino acid sequences. Certain residues that havealternative or deleted amino acid residues among V_(C)β of differentfHbps are underlined.

The variable segment V_(D) starts at residue 161, C-terminal to I₅ andends at residue 179, N-terminal to I₆. V_(D) of an α progenitor (V_(D)α)comprises an amino acid sequence that is about 89 to 100% identical toSegment ID D.α.1 as presented in FIG. 2D (Table 8): AGGKLTYTID FAAKQGHGK(SEQ ID NO:174). V_(D)α is further characterized by signature amino acidresidues AG or AS, of which AG is bolded in sequence. Certain residuesthat have alternative or deleted amino acid residues among V_(D)α ofdifferent fHbps are underlined.

V_(D) of a β progenitor (V_(D)β) comprises an amino acid sequence thatis about 84 to 100% identical to Segment ID D.β.1 as presented in FIG.2D (Table 8): PNGRLHYSID FTKKQGYGR (SEQ ID NO:192). V_(D)β is furthercharacterized by signature amino acid residues PN, bolded in sequence.Certain residues that have alternative or deleted amino acid residuesamong V_(D)β of different fHbps are underlined.

The variable segment V_(E) starts around residue 185, C-terminal to I₆and ends at residue 253, N-terminal to I₇. V_(E) of an α progenitor(V_(E)α) comprises an amino acid sequence that is about 86 to 100%identical to Segment ID E.α.1 as presented in FIG. 2E (Table 8):SPELNVDLAA AYIKPDEKHH AVISGSVLYN QAEKGSYSLG IFGGKAQEVA GSAEVKTVNGIRHIGLAA (SEQ ID NO:195). V_(E)α is further characterized by signatureamino acid residues SLGI, bolded in sequence. Certain residues that havealternative or deleted amino acid residues among V_(E)α of differentfHbps are underlined.

V_(E) of a β progenitor (V_(E)β) comprises an amino acid sequence thatis about 94 to 100% identical to Segment ID E.β.1 as set forth in FIG.2E (Table 8): TPEQNVELAS AELKADEKSH AVILGDTRYG GEEKGTYHLA LFGDRAQEIAGSATVKIREK VHEIGIAG (SEQ ID NO:262). V_(E)β is further characterized bysignature amino acid residues HLAL, bolded in sequence. Certain residuesthat have alternative or deleted amino acid residues among V_(E)β ofdifferent fHbps are underlined.

As noted above, for convenience and clarity residue numbering used inreference to non-naturally occurring chimeric fHbps throughout thepresent disclosure is based on the amino acid sequence numbering of thefHbp of MC58 (peptide ID 1). In addition, the residue position listedabove at which each variable segment starts and ends may be shifted from1 to 8 residues depending on the length of the N-terminal element andthe amino acid sequence of the variable segments.

fHbp Modular Groups

Naturally occurring fHbp amino acid sequences can be described accordingto a modular architecture based on a combination of α or β progenitorsegments flanked by invariable segments, as shown in FIG. 1A-1G (Table7) and FIGS. 10A-10I (Table 9). As described in the Examples below,naturally occurring fHbps exhibit particular combinations of modularsegments, and can be classified accordingly into nine modular grouptypes as shown in FIG. 8, where gray variable segments correspond to theα progenitor sequences and white segments correspond to the β progenitorsequences.

The Type I modular group is characterized by variable segments, all ofwhich are derived from the α progenitor amino acid sequences. FHbpsclassified as belonging to the Type I modular group may be representedby the following formula:I₁-Nte-I₂-V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)α-I₆-V_(E)α-I₇. An exampleof a fHbp that is of the Type I modular group is peptide ID 1 fromstrain MC58. As noted above, peptide ID is from the factor H-bindingprotein database at Neisseria.org.

The Type II modular group is characterized by variable segments, all ofwhich are derived from the β progenitor. FHbps classified as belongingto the Type II modular group may be represented by the followingformula: I₁-Nte-I₂-V_(A)β-I₃-V_(B)β-I₄-V_(C)β-I₅-V_(D)β-I₆-V_(E)β-I₇. Anexample of a fHbp that is of the Type II modular group is peptide ID 28from strain M1239. Nearly 60% of the fHbps in FIG. 1A-1G (Table 7)belong in either Type I or II modular group.

Some fHbp, however, have variable segments from both α and βprogenitors. One modular group defined by variable segments derived fromboth progenitors is Type III, in which variable segments V_(A) and V_(B)are derived from the α progenitor and the rest of the variable segmentsfrom the β progenitor. FHbps classified as belonging to the Type IIImodular group may be represented by the following formula:I₁-Nte-I₂-V_(A)α-I₃-V_(B)α-I₄-V_(C)β-I₅-V_(D)β-I₆-V_(E)β-I₇. One exampleof a fHbp that belongs to the Type III modular group is peptide ID 22from strain RM1090.

Another modular group defined by variable segments derived from bothprogenitors is Type IV. Type IV is a modular group that has a variablesegment V_(A) derived from the β progenitor and the rest of the variablesegments from the α progenitor. FHbps classified as belonging to theType IV modular group may be represented by the following formula:I₁-Nte-I₂-V_(A)β-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)α-I₆-V_(E)α-I₇. One exampleof a fHbp that belongs to the Type IV modular group is peptide ID 15from strain NM452.

An additional modular group is Type V, in which the variable segmentV_(D) is derived from the α progenitor and the rest of the variablesegments from the β progenitor. FHbps classified as belonging to theType V modular group may be represented by the following formula:I₁-Nte-I₂-V_(A)β-I₃-V_(B)β-I₄-V_(C)β-I₅-V_(D)α-I₆-V_(E)β-I₇. One exampleof a fHbp that belongs to the Type V modular group is peptide ID 79 fromstrain S3032.

Another modular group set forth in FIG. 8 is Type VI, in which variablesegments V_(C) and V_(E) are derived from the β progenitor and the restof the variable segments from the α progenitor. FHbps classified asbelonging to the Type VI modular group may be represented by thefollowing formula:I₁-Nte-I₂-V_(A)α-I₃-V_(B)α-I₄-V_(C)β-I₅-V_(D)α-I₆-V_(E)β-I₇. One exampleof a fHbp that belongs to the Type VI modular group is peptide ID 16from strain 961-5945.

Another modular group set forth in FIG. 8 is Type VII, in which variablesegment V_(E) are derived from the β progenitor and the rest of thevariable segments from the α progenitor. FHbps classified as belongingto the Type VI modular group may be represented by the followingformula: I₁-Nte-I₂-V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)α-I₆-V_(E)β-I₇.One fHbp found to have the Type VI modular group is peptide ID 207 fromstrain 0167/03.

Another modular group set forth in FIG. 8 is Type VIII, in whichvariable segments V_(B) is derived from the α progenitor and the rest ofthe variable segments from the β progenitor. FHbps classified asbelonging to the Type VI modular group may be represented by thefollowing formula:I₁-Nte-I₂-V_(A)β-I₃-V_(B)α-I₄-V_(C)β-I₅-V_(D)β-I₆-V_(E)β-I₇. One fHbpfound to have the Type VIII modular group is peptide ID 67 from strainMA-5756.

The last modular group set forth in FIG. 8 is Type IX, in which variablesegments V_(B) and V_(D) are derived from the α progenitor and the restof the variable segments from the β progenitor. FHbps classified asbelonging to the Type VI modular group may be represented by thefollowing formula:I₁-Nte-I₂-V_(A)β-I₃-V_(B)α-I₄-V_(C)β-I₅-V_(D)α-I₆-V_(E)β-I₇. One Exampleof a fHbp that belongs to the Type IX modular group is peptide ID 175from strain 19498.

More than 98% of the unique fHbp amino acid sequences analyzed hereinbelong to one of the first six types of modular groups (I, II, III, IV,V, and VI) described above and schematically presented in FIG. 8. Thenumbers of unique fHbp that belong to each of the modular groups arealso listed on the right of the schematic in FIG. 8.

Four naturally occurring fHbps vary at least slightly from thisnomenclature, and do not precisely fit into the first six modular grouptypes set forth in FIG. 8, panel A. These four fHbps “switch” from oneprogenitor amino acid sequence to another at a residue position that iswithin a canonical variable segment as described above. As a result,they each contain a heterologous variable segments and their junctionpoints deviate from junction points of the rest of the fHbps found to beat I₃, I₄, I₅, or I₆ defined above. See FIG. 8, panel B for the modulararchitecture of these four fHbps along with the arrow pointing at thejunction point for each. However, the junction points of these fournaturally occurring fHbps still reside at conserved residue(s) foundwithin the variable segment. See table in FIG. 8, panel C.

One such fHbp is a naturally occurring fHbp derived from strain CDC-1573(peptide ID 55), in which the junction point where V_(A)β switches toV_(A)α resides in the middle of V_(A). The heterologous V_(A) of peptideID 55 may be represented as V_(A)(β→α), signifying that the variablesegment A comprises a β progenitor amino acid sequence starting at theN-terminus and switches to an α progenitor amino acid sequence towardthe C-terminus. The junction point (J₁) of peptide ID 55 resides at asequence of AQGAE (SEQ ID NO:278) conserved among all the V_(A)sequences, which starts at residue position 50. Two other fHbps, whichcontain junction points that are not in the residue position of I₃, I₄,I₅, or I₆, are peptide ID 24 and peptide ID 25, derived from strain M08240117 and strain 6275, respectively. Their junction points (J₂) atwhich V_(B)α switches to V_(B)β reside in the middle of V_(B) (residue82), at which there is an amino acid sequence of IEV) conserved amongall V_(B)s. The V_(B) of peptide ID 24 and 25 may be represented asV_(B)(α→β). Lastly, peptide ID 82 from strain MD1475 switches fromV_(E)α to V_(E)β at residue A196 (J₃), which is conserved among allV_(E). The variable segment E of peptide ID 82 may be represented asV_(E)(α→β).

As will be discussed in detail below, chimeric fHbps can be constructedusing the invariant and variable segments of this modular architectureto produce amino acid sequences not found in nature.

Nucleic Acids Encoding fHbp Segments

fHbp polypeptides, and encoding nucleic acids, from which segments ofthe chimeric fHbps of the present disclosure can be derived may be fromany suitable N. meningitidis strain. As is known in the art, N.meningitidis strains are divided into serologic groups (capsulargroups), serotypes (PorB phenotypes) and subtypes (PorA phenotypes) onthe basis of reactions with polyclonal (Frasch, C. E. and Chapman, 1973,J. Infect. Dis. 127: 149-154) or monoclonal antibodies that interactwith different surface antigens. Capsular grouping traditionally hasbeen based on immunologically detectable variations in the capsularpolysaccharide but is being replaced by PCR of genes encoding specificenzymes responsible for the biosynthesis of the structurally differentcapsular polysaccharides. About 12 capsular groups (including A, B, C,X, Y, Z, 29-E, and W-135) are known. Strains of the capsular groups A,B, C, Y and W-135 account for nearly all meningococcal disease.Serotyping traditionally has been based on monoclonal antibody definedantigenic differences in an outer membrane protein called Porin B(PorB). Antibodies defining about 21 serotypes are currently known(Sacchi et al., 1998, Clin. Diag. Lab. Immunol. 5:348). Serosubtypinghas been based on antibody defined antigenic variations on an outermembrane protein called Porin A (PorA). Both serotyping andserosubtyping are being replaced by PCR and/or DNA sequencing foridentification of genes encoding the variable regions of PorB and PorA,respectively that are associated with mAb reactivity (e.g. Sacchi, Lemoset al., supra; Urwin et al., 1998, Epidem. and Infect. 120:257).

While N. meningitidis strains of any capsular group may be used, N.meningitidis strains of capsular group B are of particular interest assources from which nucleic acid encoding fHbp and domains thereof arederived.

While the specification provides the amino acid sequence of examples offHbps from which the chimeric fHbp can be derived, this is not intendedto be limiting. For example, the chimeric fHbp can contain amino acidsequences that are at least 80%, at least 85%, at least 90%, or at least95% identical to an amino acid sequence of a naturally occurring fHbp.

Nucleic acids encoding fHbp polypeptides for use in construction ofchimeric fHbps contemplated herein are known in the art. Examples offHbp polypeptides are described in, for example, WO 2004/048404;Masignani et al. 2003 J Exp Med 197:789-799; Fletcher et al. InfectImmun 2004 2088-2100; Welsch et al. J Immunol 2004 172:5606-5615; and WO99/57280. Nucleic acid (and amino acid sequences) for fHbp variants andsubvariants are also provided in GenBank as accession nos.:NC_(—)003112, GeneID: 904318 (NCBI Ref. NP_(—)274866), peptide ID 1 fromN. meningitidis strain MC58; AY548371 (AAT01290.1) (from N. meningitidisstrain CU385); AY548370 (AAT01289.1) (from N. meningitidis strainH44/76); AY548377 (AAS56920.1) peptide ID 4 from N. meningitidis strainM4105; AY548376 (AAS56919.1) (from N. meningitidis strain M1390);AY548375 (AAS56918.1) (from N. meningitidis strain NZ98/254); AY548374(AAS56917.1) (from N. meningitidis strain M6190); AY548373 (AAS56916.1)(from N. meningitidis strain 4243); and AY548372 (AAS56915.1) (from N.meningitidis strain BZ83).

For purposes of identifying relevant amino acid sequences contemplatedfor use in the chimeric fHbps disclosed herein, it should be noted thatthe immature fHbp protein includes a leader sequence of about 19residues. Furthermore, when provided an amino acid sequence theordinarily skilled person can readily envision the sequences of nucleicacid that can encode for, and provide for expression of, a polypeptidehaving such an amino acid sequence.

In addition to the specific amino acid sequences and nucleic acidsequences provided herein, the disclosure also contemplates polypeptidesand nucleic acids having sequences that are at least 80%, at least 85%,at least 90%, or at least 95% identical in sequence to the amino acidand nucleic acids. The terms “identical” or percent “identity,” in thecontext of two or more polynucleotide sequences, or two or more aminoacid sequences, refers to two or more sequences or subsequences that arethe same or have a specified percentage of amino acid residues ornucleotides that are the same (e.g., at least 80%, at least 85%, atleast 90%, or at least 95% identical over a specified region), whencompared and aligned for maximum correspondence over a designatedregion, e.g., a V_(E) or a region at least about 40, 45, 50, 55, 60, 65or more amino acids or nucleotides in length, and can be up to thefull-length of the reference amino acid or nucleotide sequence (e.g., afull-length fHbp). The disclosure specifically contemplates bothnaturally-occurring polymorphisms and synthetically produced amino acidsequences and their encoding nucleic acids.

For sequence comparison, typically one sequence acts as a referencesequence (e.g., a naturally-occurring fHbp polypeptide sequence), towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are input into a computerprogram, sequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. The sequence comparisonalgorithm then calculates the percent sequence identity for the testsequence(s) relative to the reference sequence, based on the designatedprogram parameters.

Examples of algorithms that are suitable for determining percentsequence identity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 andAltschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov).Further exemplary algorithms include ClustalW (Higgins D., et al. (1994)Nucleic Acids Res 22: 4673-4680), available atwww.ebi.ac.uk/Tools/clustalw/index.html.

In one embodiment, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine.

Sequence identity between two nucleic acids can also be described interms of hybridization of two molecules to each other under stringentconditions. The hybridization conditions are selected following standardmethods in the art (see, for example, Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,N.Y.). An example of stringent hybridization conditions is hybridizationat 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodiumcitrate). Another example of stringent hybridization conditions isovernight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Stringent hybridization conditions are hybridizationconditions that are at least as stringent as the above representativeconditions, where conditions are considered to be at least as stringentif they are at least about 80% as stringent, typically at least 90% asstringent as the above specific stringent conditions.

The chimeric fHbps of the present disclosure are described in moredetail below in the context of modular architecture described above.

Chimeric Factor H Binding Proteins

The chimeric fHbp of the present disclosure refers to non-naturallyoccurring chimeric fHbp, including fHbps that may be described by amodular architecture as those set forth in FIG. 8, panels A and B and amodular architecture not shown in FIG. 8, panels A and B. The term“chimeric factor H binding protein” or “chimeric fHbp” refers to apolypeptide comprising, from N-terminus to C-terminus, a group of fivevariable segments represented by V_(A), V_(B), V_(C), V_(D), and V_(E),in which the combination of α/β progenitor segments and/or combinationof alleles of which is not found in nature. One example of the chimericfHbps of the present disclosure can contain at least one or more of thevariable segments derived from a different progenitor than at least oneother variable segment in the chimeric fHbp. In other words, thechimeric fHbp contains at least one variable segment of an α progenitoramino acid sequences and at least one variable segment of a βprogenitor, such that the combination is not found in nature. In anotherexample, the fHbp may also be of a modular architecture shown in FIG. 8,panel A (e.g. modular I or II in which all variable segments are α or β,respectively), in which each segment is of an allele such that thecombination of the alleles is not found in nature.

The variable segments may be flanked by invariant segments (I₂, I₃, I₄,I₅, I₆, I₇), which can act as linkers for the variable segments.Accordingly, the chimeric fHbps of the present disclosure can bedescribed by the formula:

V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)

wherein each of the variable segments (V_(A), V_(B), V_(C), V_(D), andV_(E)) can be derived from either an α or a β progenitor amino acidsequence.

Chimeric fHbp can optionally include a leader sequence, e.g., to providefor expression of the chimeric fHbp on a cell surface of a bacterialhost cell Chimeric fHbp may also include about 1, 5, 10, or moreresidues as an N-terminal element (Nte) and/or C-terminal element (Cte).For example, the Nte may comprise 1, 2, or 6 glycine residues, or about6 residues of a mixture of glycines and serines following the N-terminalmost invariant segment (I₁).

In addition, chimeric fHbps of the present disclosure can optionallyinclude an N-terminal element, one or both of invariant segments thatflank the N-terminal element, and/or most C-terminal invariant segment(I₇). A chimeric fHbp comprising one or more of these optional featurescan be described by the following formula:

I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E);

Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E);

I₁-Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E);

V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-I₇;

I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-I₇;

Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-I₇; and

I₁-Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-I₇.

in which the variable segments (V_(A), V_(B), V_(C), V_(D), and V_(E))can be derived from either an α or β progenitor amino acid sequence.

In addition to having various combinations of variable segments, eachderived from either an α or β progenitor amino acid sequence, chimericfHbp of the present disclosure may also contain one or more variablesegments that are heterologous with respect to α and β progenitor aminoacid sequences. A heterologous variable segment refers to a variablesegment that comprises both a contiguous α and a contiguous β progenitoramino acid sequences, with the point at which the amino acid sequence“switches” to the other progenitor sequence referred to herein as a“junction point”. E.g. See V_(A) of peptide ID 55, V_(B) of peptide ID24 and ID 25, and V_(E) of peptide ID 82. Particularly, one or more ofthe junction points (J₁, J₂, or J₃) within a variable segment describedabove for peptide ID 55, 24, 25, and 82 may be used to construct achimeric fHbp having a heterologous variable segment. See table in FIG.8, panel C for the conserved residues in each variable segment thatserve as junction points.

Variable segments of the non-naturally occurring chimeric fHbps of thepresent disclosure may have an amino acid sequence of at least 80%, atleast 85%, at least 90%, at least 95% identical to a correspondingsegment in a naturally occurring fHbp, and can be 100% identical inamino acid sequence.

Examples of chimeric fHbps include those containing one or more of thefollowing variable segments. Each segment can be independently selectedfrom any alleles in the corresponding segment shown in FIGS. 2A-2E,Table 8. The variable segments are described in more detail below usingcertain alleles as examples.

V_(A) contains a contiguous amino acid sequence that is at least 85%, atleast 90%, or at least 95% identical to Segment ID A.α.1 or A.β.1 (SEQID NO:15 or NO:67) shown in FIG. 2A (Table 8), e.g., at least 85%, atleast 90%, or at least 95% identical to one of the amino acid sequencesshown in FIG. 2A (Table 8) for Segment A.

V_(B) contains a contiguous amino acid sequence that is at least 75%, atleast 80%, at least 90%, or at least 95% identical to Segment ID B.α.1or B.β.1 (SEQ ID NO:85 or NO:95) shown in FIG. 2B (Table 8), e.g., atleast 75%, at least 80%, at least 90%, or at least 95% identical to oneof the amino acid sequences shown in FIG. 2B (Table 8) for Segment B.

V_(C) contains s a contiguous amino acid sequence that is at least 80%,at least 85%, at least 90%, or at least 95% identical to Segment IDC.α.1 or C.β.1 (SEQ ID NO:96 or NO:152) shown in FIG. 2C (Table 8),e.g., at least 80%, at least 85%, at least 90%, or at least 95%identical to one of the amino acid sequences shown in FIG. 2C (Table 8)for Segment C.

V_(D) contains a contiguous amino acid sequence that is at least 80%, atleast 85%, at least 90%, or at least 95% identical to Segment ID D.α.1or D.β.1 (SEQ ID NO:174 or NO:192) shown in FIG. 2D (Table 8), e.g., atleast 80%, at least 85%, at least 90%, or at least 95% identical to oneof the amino acid sequences shown in FIG. 2D (Table 8) for Segment D.

V_(E) contains a contiguous amino acid sequence that is at least 80%, atleast 85%, at least 90%, or at least 95% identical to Segment ID E.α.1or E.β.1 (SEQ ID NO:195 or NO:262) shown in FIG. 2E (Table 8), e.g., atleast 80%, at least 85%, at least 90%, or at least 95% identical to oneof the amino acid sequences shown in FIG. 2E (Table 8) for Segment E.

The variable segments in the chimeric fHbp of the present disclosure mayalso be of the similar length as the corresponding segments listed inFIG. 2A-2E (Table 8). For example, a variable segment may be no morethan about 50, 30, 20, 10, 5, or 1 amino acid residues less or more thanone of the corresponding amino acid sequences shown in FIG. 2A-2E (Table8).

The chimeric fHbps of the present disclosure have a full length aminoacid sequence that is not found in a naturally occurring fHbp. Most ofthe naturally occurring fHbps which contain only α or only β progenitorsequences or combinations thereof fall into one of the following modulargroups: I, II, III, IV, V, VI, VII, VIII, or IX (see FIG. 8).Non-naturally occurring fHbps of the present disclosure can also be of amodular group other than I, II, III, IV, V, VI, VII, VIII, or IX. Thenon-naturally occurring fHbps of the present disclosure can be describedby new modular groups encompassing non-naturally occurring amino acidsequences that are built based on the modular architecture describedherein. Some examples of chimeric fHbps of the present disclosure arepresented below.

Examples of Chimeric fHbps

The chimeric fHbps of the present disclosure encompass those that can bedescribed in terms of one or more of, for example, the invariant andvariable segments and their progenitor type, the presence or absence ofan epitope(s) specifically bound by a mAb, or any combination of suchfeatures that may be present in the chimeric fHbps.

Many combinations may be generated by switching α or β segments in andout of various segments for a different type of modular group. Anyalleles of α or β segments can also be independently chosen from thoseshown FIG. 2A-2E (Table 8). Modular group may also be designed tomaintain or include certain antibody epitopes. Certain antibodies arefound to be bactericidal against a specific variant of N. meningitidisbut not a different variant. If a chimeric fHbp of a modular groupcontains variable segments that present multiple epitopes, each specificfor an antibody bactericidal against a different variant, the chimericfHbp may be useful to elicit antibodies or immune response effectiveagainst a broad spectrum of N. meningitidis. For example, in a casewhere epitopes for JAR4, JAR3, and JAR32 antibodies are known and aredesirable to be incorporated into an fHbp, a chimeric fHbp may be madeto comprise V_(A)α, V_(C)α, and V_(D)β, where epitopes of JAR4, JAR3,and JAR32 reside, respectively.

For example, a chimeric fHbp may have V_(D) that is derived from βprogenitor sequences while the rest of the variable segments remain asderivations from α progenitor sequences. Such chimeric fHbp comprisesvariable segments represented as V_(A)α, V_(B)α, V_(C)α, V_(D)β, andV_(E)α; and its first and second junction points are at I₅ and I₆,respectively. In a different example, an fHbp belonging to anothermodular group encompassing non-naturally occurring chimeric fHbp maycontain V_(A)α, V_(B)α, V_(C)α, V_(D)β, and V_(E)β; and its junctionpoint resides at I₅. An additional example of a modular groupencompasses non-naturally occurring fHbp that comprises V_(A)α, V_(B)α,V_(C)α, V_(D)α, and V_(E)β as the invariable segments.

Accordingly, examples of non-naturally occurring chimeric fHbpsincluding those described above may be of modular groups represented bythe following formula but not limited to:

V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)β-I₆-V_(E)α;

V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)β-I₆-V_(E)β;

V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)α-I₆-V_(E)β;

V_(A)α-I₃-V_(B)α-L₄-V_(C)β-I₅-V_(D)-I₆-V_(E)α;

V_(A)α-I₃-V_(B)β-I₄-V_(C)-I₅-V_(D)-I₆-V_(E);

V_(A)β-I₃-V_(B)α-I₄-V_(C)α-15-V_(D)α-I₆-V_(E)β;

V_(A)β-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)β-I₆-V_(E)α;

V_(A)β-I₃-V_(B)α-I₄-V_(C)β-I₅-V_(D)-I₆-V_(E);

V_(A)β-I₃-V_(B)β-I₄-V_(C)β-I₅-V_(D)α-I₆-V_(E)α; or

V_(A)β-I₃-V_(B)β-I₄-V_(C)β-I₅-V_(D)β-I₆-V_(E)α;

In any group shown above in which a variable segment (V_(A), V_(B),V_(C), V_(D), or V_(E)) is not followed by an α or a β (e.g. V_(C),V_(D), and V_(E) in the fifth modular group from the top) represents avariable segment that can be derived from either an α or a β progenitoramino acid sequence. The formula may also include a variable N-terminalelement, its flanking invariable sequences (I₁ and I₂), or the mostC-terminal invariable segment (I₇), omitted in the formula above. Asnoted above for fHbp of the present disclosure, each variable segment ofthe fHbps described by the formulas above can be of any allele.

In another example, the chimeric fHbp of the present disclosure alsoencompass those that contain all α or all β segments, in which each ofthe segments is of an allele such that the combination of all α or all βallelic segments is not found in nature.

Chimeric fHbp Comprising Heterologous Variable Segment

In certain cases, non-naturally occurring fHbp of the present disclosuremay comprise one or more heterologous variable segments. Theseheterologous variable segment-containing fHbps may also be described bymodular architectures other than those set forth in FIG. 8, panel A.Thus, for example, non-naturally occurring fHbp of the presentdisclosure include those having: a heterologous V_(A)(β→α), but do nothave α progenitor sequence for all of V_(B), V_(C), V_(D), and V_(E); aheterologous V_(B)(α→β), but do not have a V_(A) of α progenitorsequence together with V_(C), V_(D), and V_(E) of β progenitor sequence;and a heterologous V_(E)(α→β), but do not have β progenitor sequence forall of V_(A), V_(B), and V_(C) together with a progenitor sequence forV_(D). Exemplary non-naturally occurring fHbps are presented by severalexamples below, each containing one heterologous variable segment.However, non-naturally occurring fHbps are not limited to the examples,e.g. an fHbp may comprise more than one heterologous variable segment.

For example, a heterologous variable segment-containing fHbp may be onecomprising a heterologous V_(A), such as V_(A)(β→α) as exemplified bypeptide ID 55, in which not all of V_(B), V_(C), V_(D), and V_(E) are ofthe α progenitor amino acid sequences. A non-naturally occurring fHbpmay also comprise V_(A)(α→β), and in which V_(B), V_(C), V_(D), andV_(E) are independently selected from α and β. Another exemplarynon-naturally occurring fHbp may comprise V_(B)(α→β) as exemplified bypeptide ID 24, in which V_(A) is of a α progenitor and not all of V_(C),V_(D), and V_(E) are of the β progenitor amino acid sequences. Anon-naturally occurring fHbp may comprise V_(B)(α→β), in which V_(A) isof a p progenitor. A non-naturally occurring fHbp may compriseV_(B)(β→α) in which V_(A), V_(C), V_(D), and V_(E) are independentlyselected from α and β. Additionally, a non-naturally occurring fHbp maycomprise a heterologous V_(E), such as V_(E)(α→β) seen in peptide ID 82,together with V_(D)α, in which not all of V_(A), V_(B), V_(C) are of theβ progenitor origin. A non-naturally occurring fHbp may compriseV_(E)(α→β) together with V_(D)β, in which V_(A), V_(B), and V_(C) areindependently selected from α and β. Another example of a non-naturallyoccurring fHbp that contains a heterologous variable segment is one thatcomprises V_(E)(β→α), in which V_(A), V_(B), V_(C), and V_(D) areindependently selected from α and β.

Chimeric fHbp Having Modified Epitope(s)

The chimeric fHbp of the present disclosure may comprise one or morevariable segments in which one or more residues may be mutated relativeto the amino acid sequence found in the naturally occurring fHbp so asto introduce an epitope of interest. These epitopes are referred toherein “heterologous epitopes” since they are heterologous to thevariable segment in which they reside. Site-directed mutagenesis may beused to introduce one or more heterologous epitopes for a specificantibody of interest into a variable segment that naturally does notcontain such epitope. As such, mutations to insert, delete, orsubstitute one or more amino acid residues may be used to createchimeric fHbp of the present disclosure. Various mutations may be alsoscreened for an fHbp that is effective as vaccines or may be immunogenicto elicit bactericidal antibodies.

For example, an fHbp may comprise an epitope that binds to an antibodyJAR13, normally found in V_(E) of a β progenitor sequence and not in anα progenitor sequence. To generate a fHbp that includes V_(E)α while atthe same time contains the JAR13 epitope, the epitope may be introducedinto a V_(E)α sequence. Selective mutation allows the amino acidsequence of V_(E) to be derived from the α progenitor type except for aspecific site for the JAR13 epitope. As such, regardless of the aminoacid sequence used to derive the sequences of the variable segments ofthe chimeric fHbp, epitopes may be introduced into the correspondingvariable segment to include or maintain regions of desired antigenicity.

Chimeric fHbps also encompass chimeric fHbp that contain two or moreepitopes that elicit antibodies that, when both are bound to theirrespective epitopes, exhibit enhanced bactericidal activity against N.meningitidis than when either one is bound alone. For example, thecombination of JAR 4 and JAR 5 mAbs exhibit higher level of bactericidalactivity against N. meningitidis than when each is used alone in ahuman-complement-mediated assay Chimeric fHbp can be designed so as toensure that such epitopes are maintained or to introduce suchcombination of epitopes Chimeric fHbps can also be designed to includean epitope(s) that elicits antibodies that, when bound to fHbp, inhibitfH binding. For example, when the epitopes bound by the monoclonalantibodies JAR3, JAR 5, JAR11, or JAR32/35 are bound by antibody,binding of fHbp to fH is inhibited. Thus, the presence of suchfH-binding epitopes in the chimeric fHbp polypeptides can provide forproduction of antibodies that can facilitate protection through thispathway.

The antibodies that bind epitopes that may be of interest to introduceor maintain in the segments of a chimeric fHbp are disclosed in WO09/114,485, the disclosure of which is incorporated in its entirety byreference. Certain epitopes of interest with a corresponding JAR mAbsare presented in the table 1 below and pointed out in FIG. 9, panel C.

TABLE 1 Antibodies and their corresponding epitopes. (Immunogen)Antibody Reactive Residue(s) fHbp variant bound mAb 502 R204 1 JAR 3G121 and K122 1 JAR 4 DHK (starting at 25); v.1, v.2 (high reactivity)YGN (starting at 57) and v.3 (lower reactivity) JAR 5 G121 and K122 v.1JAR 10 K180 and E192 v.1 (subset), v.2 (subset) and v. 3 (subset) JAR 11A174 v.2 (subset) and v.3 (subset) JAR 13 S216 v. 2 (subset) and v.3(all) JAR 32 K174 v. 3 and v.2 (subset) JAR 33 E180 and R192 v. 3 andv.2 (subset) JAR 35 K174 v. 3 and v.2 (subset)

Other Features of Interest

Chimeric polypeptides described herein can include additionalheterologous amino acid sequences, e.g., to provide an N-terminalmethionine or derivative thereof (e.g., pyroglutamate) as a result ofexpression in a bacterial host cell (e.g., E. coli) and/or to provide achimeric polypeptide having a fusion partner at its N-terminus orC-terminus. Fusion partners of interest include, for example,glutathione-S-transferase (GST), maltose binding protein (MBP),His₆-tag, and the like, as well as leader peptides from other proteins,particularly lipoproteins. Fusion partners can provide for additionalfeatures, such as in facilitating isolation and purification of thechimeric polypeptide.

Native fHbp usually contains an N-terminal cysteine to which a lipidmoiety can be covalently attached. This cysteine residue is usuallylipidated in the naturally-occurring protein, and can be lipidated inthe chimeric fHbps disclosed herein. Thus, in the amino acid sequencesdescribed herein (including those presented in any Sequence Listing),reference to “cysteine” or “C” at this position specifically includesreference to both an unmodified cysteine as well as to a cysteine thatis lipidated (e.g., due to post-translational modification). Thus, thechimeric fHbp can be lipidated or non-lipidated. Methods for productionof lipidated proteins in vitro, (see, e.g., Andersson et al., 2001 JImmunological Methods 255(1-2):135-48) or in vivo are known in the art.For example, lipidated fHbp previously has been purified from themembrane fraction of E. coli protein by detergent extraction (Fletcheret al., 2004 Infection and Immunity 72(4):2088-100), which method may beadapted for the production of lipidated chimeric fHbp. Lipidatedproteins may be of interest as such can be more immunogenic than solubleprotein (see, e.g., Fletcher et al., 2004 Infection and Immunity72(4):2088-100).

Nucleic Acid Encoding Chimeric fHBP

The chimeric fHbp can be generated using recombinant techniques tomanipulate nucleic acids of different fHbps known in the art to provideconstructs encoding a chimeric fHbp of interest. As noted above, nucleicacids encoding a variety of different v.1, v.2, and v.3 fHbps of N.meningitidis are available in the art, and their nucleotide sequencesare known.

Amino acid and nucleic acid sequences of the naturally occurring fHbpsare provided in FIG. 2A-2E (Table 5) and available in the art. It willbe appreciated that the nucleotide sequences encoding the chimeric fHbpscan be modified so as to optimize the codon usage to facilitateexpression in a host cell of interest (e.g., E. coli, N. meningitidis,human (as in the case of a DNA-based vaccine), and the like). Methodsfor production of codon optimized sequences are known in the art.

Methods of Screening

The present disclosure also features methods of screening for animmunogenic composition comprising a chimeric fHbp, vaccine againstbacterial pathogens, antibodies and nucleic acid encoding the same. Themethods may involve evaluating the ability of a specific chimeric fHbpto elicit bactericidal antibodies and/or to provide a broad spectrumimmunity against bacterial pathogens. The methods can involve assessingthe ability of antibodies elicited by immunization with a chimeric fHbpof the present disclosure to inhibit fH binding to fHbp of N.meningitidis of diverse variant groups and/or to elicit bactericidalactivity. The antibody may be elicited in a host animal immunized withan immunogenic composition comprising a chimeric fHbp of the presentdisclosure or screened in a phage display library for its specificaffinity to a candidate chimeric fHbp. Among other aspects, the methodsfind particular use in identifying and/or evaluating antibody havingbactericidal and/or anti-neoplastic activity.

In one embodiment, a method of evaluating binding of an antibody to abacterial cell is provided. The method comprises: (a) immunizing a hostanimal with a composition comprising a chimeric fHbp of the presentdisclosure, (b) isolating antibodies from the host animal that havebinding affinity to the chimeric fHbp, (c) contacting a bacterial cellwith the isolated antibodies; and (d) assessing binding of the antibodyto the bacterial cell. Additional steps may include assessing thecompetitive binding of the antibody to fHbp with human factor H;assessing the bactericidal activity against a bacterial pathogen whenincubated in vitro with complement; or assessing the ability of theantibody administered to an animal to confer passive protection againstinfection. In some embodiments, the antibody is in an antibodypopulation, and the method further comprises: (c) isolating one or moreantibodies of the antibody population that bind the bacterial cell. Afeatured aspect is isolated antibody that is bactericidal against thebacterial cell, which may include, for example, complement-mediatedbactericidal activity and/or opsonophagocytic activity capable ofdecreasing the viability of the bacteria in human blood. The subjectmethod may also include assessing the susceptibility of a host animaladministered with a vaccine comprising a chimeric fHbp to a bacterialpathogen.

Bacterial pathogen of particular interest are N. meningitidis of any orall fHbp variant groups, of diverse capsular groups, such as N.meningitidis Serogroup B, N. meningitidis Serogroup C, N. meningitidisSerogroup X, N. meningitidis Serogroup Y, N. meningitidis SerogroupW-135, and the like.

Methods of Production

Chimeric fHbps can be produced by any suitable method, includingrecombinant and non-recombinant methods (e.g., chemical synthesis).Where the chimeric fHbp is produced using recombinant techniques, themethods can involve any suitable construct and any suitable host cell,which can be a prokaryotic or eukaryotic cell, usually a bacterial oryeast host cell, more usually a bacterial cell. Methods for introductionof genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced chimeric fHbp-encoding nucleicacid. The chimeric fHbp-encoding nucleic acid can be provided as aninheritable episomal element (e.g., plasmid) or can be genomicallyintegrated.

Suitable vectors for transferring chimeric fHbp-encoding nucleic acidcan vary in composition. Integrative vectors can be conditionallyreplicative or suicide plasmids, bacteriophages, and the like. Theconstructs can include various elements, including for example,promoters, selectable genetic markers (e.g., genes conferring resistanceto antibiotics (for instance kanamycin, erythromycin, chloramphenicol,or gentamycin)), origin of replication (to promote replication in a hostcell, e.g., a bacterial host cell), and the like. The choice of vectorwill depend upon a variety of factors such as the type of cell in whichpropagation is desired and the purpose of propagation. Certain vectorsare useful for amplifying and making large amounts of the desired DNAsequence. Other vectors are suitable for expression in cells in culture.Still other vectors are suitable for transfer and expression in cells ina whole animal The choice of appropriate vector is well within the skillof the art. Many such vectors are available commercially.

The vector can be an expression vector based on episomal plasmidscontaining selectable drug resistance markers and elements that providefor autonomous replication in different host cells (e.g., in both E.coli and N. meningitidis). One example of such a “shuttle vector” is theplasmid pFP10 (Pagotto et al. Gene 2000 244:13-19).

Constructs can be prepared by, for example, inserting a polynucleotideof interest into a construct backbone, typically by means of DNA ligaseattachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination or site-specific recombination. Typicallyhomologous recombination is accomplished by attaching regions ofhomology to the vector on the flanks of the desired nucleotide sequence,while site-specific recombination can be accomplished through use ofsequences that facilitate site-specific recombination (e.g., cre-lox,att sites, etc.). Nucleic acid containing such sequences can be addedby, for example, ligation of oligonucleotides, or by polymerase chainreaction using primers comprising both the region of homology and aportion of the desired nucleotide sequence.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding a chimeric fHBP, may provide forpropagating the subject nucleic acids, or both.

Examples of vectors that may be used include but are not limited tothose derived from recombinant bacteriophage DNA, plasmid DNA or cosmidDNA. For example, plasmid vectors such as pBR322, pUC19/18, pUC118, 119and the M13 mp series of vectors may be used. pET21 is also anexpression vector that may be used. Bacteriophage vectors may includeλgt10, λgt11, λgt18-23, λZAP/R and the EMBL series of bacteriophagevectors. Further vectors that may be utilized include, but are notlimited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274,COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

For expression of a chimeric fHbp of interest, an expression cassettemay be employed. Thus, the present disclosure provides a recombinantexpression vector comprising a subject nucleic acid. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toan fHbp from which the chimeric fHbp is derived, or may be derived fromexogenous sources. In general, the transcriptional and translationalregulatory sequences may include, but are not limited to, promotersequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7, and the like).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host may be present to facilitate selection of cellscontaining the vector. In addition, the expression construct may includeadditional elements. For example, the expression vector may have one ortwo replication systems, thus allowing it to be maintained in organisms,for example in mammalian or insect cells for expression and in aprokaryotic host for cloning and amplification. In addition theexpression construct may contain a selectable marker gene to allow theselection of transformed host cells. Selection genes are well known inthe art and will vary with the host cell used.

It should be noted that chimeric fHbp of the present disclosure maycomprise additional elements, such as a detectable label, e.g., aradioactive label, a fluorescent label, a biotin label, animmunologically detectable label (e.g., an HA tag, a poly-Histidine tag)and the like. Additional elements of chimeric fHbp can be provided tofacilitate isolation (e.g., biotin tag, immunologically detectable tag)through various methods (e.g., affinity capture, etc.). Chimeric fHbpcan optionally be immobilized on a support through covalent ornon-covalent attachment.

Isolation and purification of chimeric fHbp can be accomplishedaccording to methods known in the art. For example, chimeric fHbp can beisolated from a lysate of cells genetically modified to express achimeric fHbp, or from a synthetic reaction mix, by immunoaffinitypurification, which generally involves contacting the sample with ananti-chimeric fHbp antibody (e.g., an anti-chimeric fHBP mAb, such as aJAR 5 mAb or other appropriate JAR mAb described herein), washing toremove non-specifically bound material, and eluting specifically boundchimeric fHbp. Isolated chimeric fHbp can be further purified bydialysis and other methods normally employed in protein purificationmethods. In one embodiment, the chimeric fHbp can be isolated usingmetal chelate chromatography methods.

Host Cells

Any of a number of suitable host cells can be used in the production ofchimeric fHbp. In general, the chimeric fHbp described herein may beexpressed in prokaryotes or eukaryotes, usually bacteria, more usuallyE. coli or Neisseria (e.g., N. meningitidis) in accordance withconventional techniques. Thus, the present disclosure further provides agenetically modified host cell, which contains a nucleic acid encoding achimeric fHBP. Host cells for production (including large scaleproduction) of a chimeric fHBP can be selected from any of a variety ofavailable host cells. Exemplary host cells for expression include thoseof a prokaryotic or eukaryotic unicellular organism, such as bacteria(e.g., Escherichia coli strains), yeast (e.g., S. cerevisiae, Pichiaspp., and the like), and may include host cells originally derived froma higher organism such as insects, vertebrates, particularly mammals,(e.g. CHO, HEK, and the like). Generally bacterial host cells and yeastare of particular interest for chimeric fHBP production.

Chimeric fHbps can be prepared in substantially pure or substantiallyisolated form (i.e., substantially free from other Neisserial or hostcell polypeptides) or substantially isolated form. In certainembodiments, the chimeric fHbp is present in a composition that isenriched for the polypeptide relative to other components that may bepresent (e.g., other polypeptides or other host cell components).Purified chimeric fHbp can be provided such that the polypeptide ispresent in a composition that is substantially free of other expressedpolypeptides, e.g., less than 90%, usually less than 60% and moreusually less than 50% of the composition is made up of other expressedpolypeptides.

Host Cells for Vesicle Production

Where a chimeric fHbp is to be provided in a membrane vesicle (asdiscussed in more detail below), a Neisserial host cell is geneticallymodified to express a chimeric fHbp. Any of a variety of Neisseria spp.strains can be modified to produce a chimeric fHbp, and, optionally,which produce or can be modified to produce other antigens of interest,such as PorA, can be used in the methods disclosed herein.

Methods and vectors to provide for genetic modification of Neisserialstrains and expression of a desired polypeptide are known in the art.Examples of vectors and methods are provided in WO 02/09746 and O'Dwyeret al. Infect Immun 2004; 72:6511-80. Strong promoters, particularlyconstitutive strong promoters are of particular interest. Exemplarypromoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB,lgtF, opa, p110, 1st, hpuAB. and rmp.

Pathogenic Neisseria spp. or strains derived from pathogenic Neisseriaspp., particularly strains pathogenic for humans or derived from strainspathogenic or commensal for humans, are of particular interest for usein membrane vesicle production. Examples of Neisserial spp. include N.meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N.polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N.elongata, and the like.

N. meningitidis strains are of particular interest for geneticmodification to express a chimeric fHbp and for use in vesicleproduction. The strain used for vesicle production can be selectedaccording to a number of different characteristics that may be desired.For example, the strain may be selected according to: a desired PorAtype (a “serosubtype”, as described above), capsular group, serotype,and the like; decreased capsular polysaccharide production; and thelike. For example, the production strain can produce any desired PorApolypeptide, and may express one or more PorA polypeptides (eithernaturally or due to genetic engineering). Examples of strains includethose that produce a PorA polypeptide which confers a serosubtype ofP1.7,16; P1.19,15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4;P1.12,13; as well as variants of such PorA polypeptides which may or maynot retain reactivity with conventional serologic reagents used inserosubtyping. Also of interest are PorA polypeptides characterizedaccording to PorA variable region (VR) typing (see, e.g., Russell et al.Emerging Infect Dis 2004 10:674-678; Sacchi C T, et al, Clin Diagn LabImmunol 1998; 5:845-55; Sacchi et al, J. Infect Dis 2000;182:1169-1176). A substantial number of distinct VR types have beenidentified, which can be classified into VR1 and VR2 family“prototypes”. A web-accessible database describing this nomenclature andits relationship to previous typing schemes is found atneisseria.org/nm/typing/pora. Alignments of exemplary PorA VR1 and VR2types are provided in Russell et al. Emerging Infect Dis 200410:674-678.

Alternatively or in addition, the production strain can be a capsuledeficient strain. Capsule deficient strains can provide vesicle-basedvaccines that provide for a reduced risk of eliciting a significantautoantibody response in a subject to whom the vaccine is administered(e.g., due to production of antibodies that cross-react with sialic acidon host cell surfaces). “Capsule deficient” or “deficient in capsularpolysaccharide” as used herein refers to a level of capsularpolysaccharide on the bacterial surface that is lower than that of anaturally-occurring strain or, where the strain is genetically modified,is lower than that of a parental strain from which the capsule deficientstrain is derived. A capsule deficient strain includes strains that aredecreased in surface capsular polysaccharide production by at least 10%,20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includesstrains in which capsular polysaccharide is not detectable on thebacterial surface (e.g., by whole cell ELISA using an anti-capsularpolysaccharide antibody).

Capsule deficient strains include those that are capsule deficient dueto a naturally-occurring or recombinantly-generated geneticmodification. Naturally-occurring capsule deficient strains (see, e.g.,Dolan-Livengood et al. J. Infect. Dis. (2003) 187(10):1616-28), as wellas methods of identifying and/or generating capsule-deficient strains(see, e.g., Fisseha et al. (2005) Infect. Immun. 73(7):4070-4080;Stephens et al. (1991) Infect Immun 59(11):4097-102; Frosch et al.(1990) Mol. Microbiol. 1990 4(7):1215-1218) are known in the art.

Modification of a Neisserial host cell to provide for decreasedproduction of capsular polysaccharide may include modification of one ormore genes involved in capsule synthesis, where the modificationprovides for, for example, decreased levels of capsular polysacchariderelative to a parent cell prior to modification. Such geneticmodifications can include changes in nucleotide and/or amino acidsequences in one or more capsule biosynthesis genes rendering the straincapsule deficient (e.g., due to one or more insertions, deletions,substitutions, and the like in one or more capsule biosynthesis genes).Capsule deficient strains can lack or be non-functional for one or morecapsule genes.

Of particular interest are strains that are deficient in sialic acidbiosynthesis. Such strains can provide for production of vesicles thathave reduced risk of eliciting anti-sialic acid antibodies thatcross-react with human sialic acid antigens, and can further provide forimproved manufacturing safety. Strains having a defect in sialic acidbiosynthesis (due to either a naturally occurring modification or anengineered modification) can be defective in any of a number ofdifferent genes in the sialic acid biosynthetic pathway. Of particularinterest are strains that are defective in a gene product encoded by theN-acetylglucosamine-6-phosphate 2-epimerase gene (known as synXAAF40537.1 or siaA AAA20475), with strains having this gene inactivatedbeing of especial interest. For example, in one embodiment, a capsuledeficient strain is generated by disrupting production of a functionalsynX gene product (see, e.g., Swartley et al. (1994) J. Bacteriol.176(5):1530-4).

Capsule-deficient strains can also be generated from naturally-occurringstrains using non-recombinant techniques, e.g., by use of bactericidalanti-capsular antibodies to select for strains that reduced in capsularpolysaccharide.

Where the disclosure involves use of two or more strains (e.g., toproduce antigenic compositions containing a chimeric fHbp-presentingvesicles from different strains), the strains can be selected so as todiffer in one or more strain characteristics, e.g., to provide forvesicles that differ in the chimeric fHbp used, PorA, and the like.

Preparation of Vesicles

The antigenic compositions contemplated by the present disclosuregenerally include vesicles prepared from Neisserial cells that express achimeric fHbp. As referred to herein “vesicles” is meant to encompassouter membrane vesicles as well as microvesicles (which are alsoreferred to as blebs).

The antigenic composition can contain outer membrane vesicles (OMV)prepared from the outer membrane of a cultured strain of Neisseriameningitidis spp. genetically modified to express a chimeric fHbp. OMVsmay be obtained from Neisseria meningitidis grown in broth or solidmedium culture, preferably by separating the bacterial cells from theculture medium (e.g. by filtration or by a low-speed centrifugation thatpellets the cells, or the like), lysing the cells (e.g. by addition ofdetergent, osmotic shock, sonication, cavitation, homogenization, or thelike) and separating an outer membrane fraction from cytoplasmicmolecules (e.g. by filtration; or by differential precipitation oraggregation of outer membranes and/or outer membrane vesicles, or byaffinity separation methods using ligands that specifically recognizeouter membrane molecules; or by a high-speed centrifugation that pelletsouter membranes and/or outer membrane vesicles, or the like); outermembrane fractions may be used to produce OMVs.

The antigenic composition can contain microvesicles (MV) (or “blebs”)containing chimeric fHbp, where the MV or blebs are released duringculture of a Neisseria meningitidis strain genetically modified toexpress a chimeric fHbp. For example, MVs may be obtained by culturing αstrain of Neisseria meningitidis in broth culture medium, separatingwhole cells from the broth culture medium (e.g. by filtration, or by alow-speed centrifugation that pellets only the cells and not the smallerblebs, or the like), and then collecting the MVs that are present in thecell-free culture medium (e.g. by filtration, differential precipitationor aggregation of MVs, or by a high-speed centrifugation that pelletsthe blebs, or the like). Strains for use in production of MVs cangenerally be selected on the basis of the amount of blebs produced inculture (e.g., bacteria can be cultured in a reasonable number toprovide for production of blebs suitable for isolation andadministration in the methods described herein). An example of α strainthat produces high levels of blebs is described in PCT Publication No.WO 01/34642. In addition to bleb production, strains for use in MVproduction may also be selected on the basis of NspA production, wherestrains that produce higher levels of NspA may be of particular interest(for examples of N. meningitidis strains having different NspAproduction levels, see, e.g., Moe et al. (1999 Infect. Immun. 67: 5664).Other strains of interest for use in production of blebs include strainshaving an inactivated GN33 gene, which encodes a lipoprotein requiredfor cell separation, membrane architecture and virulence (see, e.g.,Adu-Bobie et al. Infect Immun 2004; 72:1914-1919).

The antigenic compositions of the present disclosure can containvesicles from one strain, or from 2, 3, 4, 5 or more strains, whichstrains may be homologous or heterologous, usually heterologous, to oneanother. For example, the strains may be homologous or heterologous withrespect to PorA. The vesicles can also be prepared from strains thatexpress more than one chimeric fHbp (e.g., 1, 2, 3, or more chimericfHbp) which may be composed of fHbp amino acid sequences from differentvariants (v.1, v.2, or v.3) or subvariants (e.g., a subvariant of v.1,v.2, or v.3).

The antigenic compositions can contain a mixture of OMVs and MVspresenting the same or different chimeric fHbps, where the chimericfHbps may optionally present epitopes from different combinations offHbp variants and/or subvariants and where the OMVs and/or MVs may befrom the same or different strains. Vesicles from different strains canbe administered as a mixture, or can be administered serially. Theantigentic composition may contain vesicles containing one or moredifferent chimeric fHbp, in which the vesicles and the fHbp are bothderived from the same host cells. The composition can also be made fromvesicles and fHbp that are derived from different host cells so that thevesicles and fHbp are admixed after their separate purification.

Where desired (e.g., where the strains used to produce vesicles areassociated with endotoxin or particular high levels of endotoxin), thevesicles are optionally treated to reduce endotoxin, e.g., to reducetoxicity following administration. Although less desirable as discussedbelow, reduction of endotoxin can be accomplished by extraction with asuitable detergent (for example, BRIJ-96, sodium deoxycholate, sodiumlauroylsarcosinate, Empigen BB, Triton X-100, TWEEN 20 (sorbitanmonolaurate polyoxyethylene), TWEEN 80, at a concentration of 0.1-10%,preferably 0.5-2%, and SDS). Where detergent extraction is used, it ispreferable to use a detergent other than deoxycholate.

The vesicles of the antigenic compositions can be prepared withoutdetergent, e.g., without use of deoxycholate. Although detergenttreatment is useful to remove endotoxin activity, it may deplete thenative fHbp lipoprotein and/or chimeric fHbp (including lipidatedchimeric fHbp) by extraction during vesicle production. Thus it may beparticularly desirable to decrease endotoxin activity using technologythat does not require a detergent. In one approach, strains that arerelatively low producers of endotoxin (lipopolysaccharide, LPS) are usedso as to avoid the need to remove endotoxin from the final preparationprior to use in humans. For example, the vesicles can be prepared fromNeisseria mutants in which lipooligosaccharide or other antigens thatmay be undesirable in a vaccine (e.g. Rmp) is reduced or eliminated.

Vesicles can be prepared from N. meningitidis strains that containgenetic modifications that result in decreased or no detectable toxicactivity of lipid A. For example, such strain can be geneticallymodified in lipid A biosynthesis (Steeghs et al. Infect Immun 1999;67:4988-93; van der Ley et al. Infect Immun 2001; 69:5981-90; Steeghs etal. J Endotoxin Res 2004; 10:113-9; Fissha et al, Infect Immun 73:4070,2005). The immunogenic compositions may be detoxified by modification ofLPS, such as downregulation and/or inactivation of the enzymes encodedby 1pxL1 or 1pxL2, respectively. Production of a penta-acylated lipid Amade in 1pxL1 mutants indicates that the enzyme encoded by 1pxL1 addsthe C12 to the N-linked 3-OH C14 at the 2′ position of GlcN II. Themajor lipid A species found in 1pxL2 mutants is tetra-acylated,indicating the enzyme encoded by 1pxL2 adds the other C12, i.e., to theN-linked 3-OH C14 at the 2 position of GlcN I. Mutations resulting in adecreased (or no) expression of these genes (or decreased or no activityof the products of these genes) result in altered toxic activity oflipid A (van der Ley et al. 2001; 69:5981-90). Tetra-acylated (1pxL2mutant) and penta acylated (1pxL1 mutant) lipid A are less toxic thanthe wild-type lipid A. Mutations in the lipid A 4′-kinase encoding gene(1pxK) also decrease the toxic activity of lipid A. Of particularinterest for use in production of vesicles (e.g., MV or OMV) are N.meningitidis strains genetically modified so as to provide for decreasedor no detectable functional LpxL1-encoded protein. Such vesicles providefor reduced toxicity as compared to N. meningitidis strains that arewild-type for LPS production, while retaining immunogenicity of chimericfHbp.

LPS toxic activity can also be altered by introducing mutations ingenes/loci involved in polymyxin B resistance (such resistance has beencorrelated with addition of aminoarabinose on the 4′ phosphate of lipidA). These genes/loci could be pmrE that encodes a UDP-glucosedehydrogenase, or a region of antimicrobial peptide-resistance genescommon to many enterobacteriaciae which could be involved inaminoarabinose synthesis and transfer. The gene pmrF that is present inthis region encodes a dolicol-phosphate manosyl transferase (Gunn J. S.,Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. 1998.Mol. Microbiol. 27: 1171-1182).

Mutations in the PhoP-PhoQ regulatory system, which is a phospho-relaytwo component regulatory system (e.g., PhoP constitutive phenotype,PhoPc), or low Mg++ environmental or culture conditions (that activatethe PhoP-PhoQ regulatory system) lead to the addition of aminoarabinoseon the 4′-phosphate and 2-hydroxymyristate replacing myristate(hydroxylation of myristate). This modified lipid A displays reducedability to stimulate E-selectin expression by human endothelial cellsand TNF secretion from human monocytes.

Polymyxin B resistant strains are also suitable for use, as such strainshave been shown to have reduced LPS toxicity (see, e.g., van der Ley etal. 1994. In: Proceedings of the ninth international pathogenicNeisseria conference. The Guildhall, Winchester, England).Alternatively, synthetic peptides that mimic the binding activity ofpolymyxin B may be added to the antigenic compositions to reduce LPStoxic activity (see, e.g., Rustici et al. 1993, Science 259:361-365;Porro et al. Prog Clin Biol Res. 1998; 397:315-25).

Endotoxin can also be reduced through selection of culture conditions.For example, culturing the strain in a growth medium containing 0.1mg-100 mg of aminoarabinose per liter medium provides for reduced lipidtoxicity (see, e.g., WO 02/097646).

Formulations

“Antigen composition”, “antigenic composition” or “immunogeniccomposition” is used herein as a matter of convenience to refergenerically to compositions comprising a chimeric fHbp as disclosedherein, which chimeric fHbp may be optionally conjugated to furtherenhance immunogenicity. Compositions useful for eliciting antibodies,particularly antibodies against Neisseria meningitidis group B (NmB), ina human are specifically contemplated by the present disclosure.Antigenic compositions can contain 2 or more different chimeric fHbps,where the chimeric fHbps may present epitopes from differentcombinations of fHbp variants and/or subvariants.

Antigenic compositions generally comprise an immunologically effectiveamount of chimeric fHbp, and may further include other compatiblecomponents, as needed. By “immunologically effective amount” is meantthat the administration of that amount to an individual, either in asingle dose, as part of a series of the same or different antigeniccompositions, is effective to elicit an antibody response effective fortreatment or prevention of a symptom of, or disease caused by, forexample, infection by Neisseria, particularly N. meningitidis, moreparticularly Group B N. meningitidis. This amount varies depending uponthe health and physical condition of the individual to be treated, age,the capacity of the individual's immune system to produce antibodies,the degree of protection desired, the formulation of the vaccine, thetreating clinician's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.

Dosage regimen may be a single dose schedule or a multiple dose schedule(e.g., including booster doses) with a unit dosage form of the antigeniccomposition administered at different times. The term “unit dosageform,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of the antigenic compositions of the presentdisclosure in an amount sufficient to produce the desired effect, whichcompositions are provided in association with a pharmaceuticallyacceptable excipient (e.g., pharmaceutically acceptable diluent, carrieror vehicle). The antigenic composition may be administered inconjunction with other immunoregulatory agents.

Antigenic compositions can be provided in a pharmaceutically acceptableexcipient, which can be a solution such as a sterile aqueous solution,often a saline solution, or they can be provided in powder form. Suchexcipients can be substantially inert, if desired.

The antigenic compositions can further comprise an adjuvant. Examples ofknown suitable adjuvants that can be used in humans include, but are notnecessarily limited to, alum, aluminum phosphate, aluminum hydroxide,MF59 (4.3% w/v squalene, 0.5% w/v Tween 80™, 0.5% w/v Span 85),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21,MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, and the like. For experimental animals, one can useFreund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicantigen or antigenic epitope thereof.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: thesubunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining MTP-PE) formulated into submicron particles using amicrofluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components such as monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); (2) saponin adjuvants, such as QS21 or Stimulon™(Cambridge Bioscience, Worcester, Mass.) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes), whichISCOMS may be devoid of additional detergent e.g. WO 00/07621; (3)Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);(4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19,618-622; Krieg Curr opin Mol Ther2001 3:15-24; Roman et al., Nat. Med,1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Daviset al, J. Immunol, 1998, 160, 810-876; Chu et al., J. Exp. Med, 1997,186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27, 2340-2344;Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al, J Immunol, 1996, 157, 1840-1845; Cowdery et al, J Immunol,1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167, 72-78;Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al, J.Immunol., 1996, 157, 2116-2122; Messina et al, J. Immunol, 1991, 147,1759-1764; Yi et al, J Immunol, 1996, 157, 4918-4925; Yi et al, J.Immunol, 1996, 157, 5394-5402; Yi et al, J Immunol, 1998, 160,4755-4761; and Yi et al, J Immunol, 1998, 160, 5898-5906; Internationalpatent applications WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100,WO 98/55495, WO 98/37919 and WO 98/52581, i.e. containing at least oneCG dinucleotide, where the cytosine is unmethylated; (8) apolyoxyethylene ether or a polyoxyethylene ester e.g. WO 99/52549; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO 01/21152); (10) a saponin and animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO00/62800); (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO 99/11241;(13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immunostimulating agents toenhance the efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Adjuvants suitable for administration to a human are ofparticular interest.

The antigen compositions may comprise other components, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of chimeric fHbp in a formulation can vary widely(e.g., from less than about 0.1%, usually at or at least about 2% to asmuch as 20% to 50% or more by weight) and will usually be selectedprimarily based on fluid volumes, viscosities, and patient-based factorsin accordance with the particular mode of administration selected andthe patient's needs.

Chimeric fHBP-containing formulations can be provided in the form of asolution, suspension, tablet, pill, capsule, powder, gel, cream, lotion,ointment, aerosol or the like. It is recognized that oral administrationcan require protection of the compositions from digestion. This istypically accomplished either by association of the composition with anagent that renders it resistant to acidic and enzymatic hydrolysis or bypackaging the composition in an appropriately resistant carrier. Meansof protecting from digestion are well known in the art.

Chimeric fHbp-containing formulations can also be provided so as toenhance serum half-life of chimeric fHBP following administration. Forexample, where isolated chimeric fHbp are formulated for injection, thechimeric fHbp may be provided in a liposome formulation, prepared as acolloid, or other conventional techniques for extending serum half-life.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may alsobe provided in controlled release or slow-release forms.

Immunization

The chimeric fHbp-containing antigenic compositions are generallyadministered to a human subject that is at risk of acquiring aNeisserial disease so as to prevent or at least partially arrest thedevelopment of disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for therapeutic use will depend on, e.g., theantigenic composition, the manner of administration, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. Single or multiple doses of the antigeniccompositions may be administered depending on the dosage and frequencyrequired and tolerated by the patient, and route of administration.

The chimeric fHbp-containing antigenic compositions are generallyadministered in an amount effective to elicit an immune response,particularly a humoral immune response, in the host. As noted above,amounts for immunization will vary, and can generally range from about 1μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg/70 kg.Substantially higher dosages (e.g. 10 mg to 100 mg or more) may besuitable in oral, nasal, or topical administration routes. The initialadministration can be followed by booster immunization of the same ofdifferent chimeric fHbp-containing antigenic composition. Usuallyvaccination involves at least one booster, more usually two boosters.

In general immunization can be accomplished by administration by anysuitable route, including administration of the composition orally,nasally, nasopharyngeally, parenterally, enterically, gastrically,topically, transdermally, subcutaneously, intramuscularly, in tablet,solid, powdered, liquid, aerosol form, locally or systemically, with orwithout added excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

An anti-chimeric fHbp immune response can be assessed by known methods(e.g. by obtaining serum from the individual before and after theinitial immunization, and demonstrating a change in the individual'simmune status, for example an immunoprecipitation assay, or an ELISA, ora bactericidal assay, or a Western blot, or flow cytometric assay, orthe like).

The antigenic compositions can be administered to a human subject thatis immunologically naive with respect to Neisseria meningitidis. In aparticular embodiment, the subject is a human child about five years oryounger, and preferably about two years old or younger, and theantigenic compositions are administered at any one or more of thefollowing times: two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11months, or one year or 15, 18, or 21 months after birth, or at 2, 3, 4,or 5 years of age.

It may be generally desirable to initiate immunization prior to thefirst sign of disease symptoms, or at the first sign of possible oractual exposure to infection or disease (e.g., due to exposure orinfection by Neisseria).

ATCC Deposit

Hybridomas producing the JAR 4, JAR 5, JAR 11, and JAR 32 monoclonalantibodies were deposited under the terms of the Budapest Treaty withthe American Type Culture Collection, 10801 University Blvd., Manassas,Va. 20110-2209, USA (ATCC) on the date indicated in the table below, andwere assigned the designations set out in the table below.

ATCC Deposit No. (Deposit Date) Material Deposited PTA-8943 (Feb. 7,2008) Hybridoma producing JAR 4 Monoclonal Antibody PTA-8941 (Feb. 7,2008) Hybridoma producing JAR 5 Monoclonal Antibody PTA-8940 (Feb. 7,2008) Hybridoma producing JAR 10 Monoclonal Antibody PTA-8938 (Feb. 7,2008) Hybridoma producing JAR 11 Monoclonal Antibody PTA-8942 (Feb. 7,2008) Hybridoma producing JAR 32 Monoclonal Antibody PTA-8939 (Feb. 7,2008) Hybridoma producing JAR 33 Monoclonal Antibody

It should be noted that JAR 5 mAb specifically binds to an epitope thatat least overlaps with the epitope specifically bound by JAR 3 mAb, andthat JAR 32 mAb specifically binds to an epitope that at least overlapswith the epitope specifically bounds by JAR 35 mAb.

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Children's Hospital & Research Center at Oakland and the ATCC(the assignee of the present application) which assures that allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed upon the grantingof the pertinent U.S. patent, assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.14 with particular reference to 8860G 638).

The assignee(s) of the present application has agreed that if a cultureof the materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Materials and Methods

The following methods and materials were used in the Examples below.

fHbp sequencing. The fHbp gene was amplified from genomic DNA preparedwith the DNeasy Tissue kit (Qiagen, Valencia, Calif.) by polymerasechain reaction using primers A1 and B2 and cycling parameters describedpreviously (Masignani et al. J Exp Med 2003, 197:789-99). The PCRproducts were purified using QiaQuick PCR purification kit (Qiagen) andwere eluted in 30 μl of sterile deionized H₂O. The fHbp DNA sequenceswere determined by a commercial sequencing facility using the primers Aland 22 described previously (Masignani et al., 2003).

Source of data. Protein sequences analyzed in the first study wereencoded by 69 fHbp genes from Neisseria meningitidis case isolates fromthe United States (Beernink et al. J Infect Dis 2007, 195:1472-9),Europe (Beernink and Granoff Infect Immun 2008, 76:2568-2575; Beerninket al. Infect Immun 2008, 76:4232-4240) and Africa (Beernink et al., JInfect Dis 2009, doi:10.1086/597806). This data set included 48sequences determined as part of our previous studies and 21 newsequences performed for the present study. 95 additional fHbp genesequences were obtained from Genbank (www.ncbi.nlm.nih.gov) byperforming translated BLAST (tblastn) searches with fHbp amino acidsequences from strains MC58 (variant 1/sub-family B) and M1239 (variant3/sub-family A). Among these 164 nucleotide sequences from ourcollection and Genbank, which included three genome sequences (Parkhillet al. Nature 2000, 404:502-506; Peng et al. Genomics 2008, 91:78-87;Tettelin et al. Science 2000, 287:1809-1815), fHbp genes that encoded 63unique protein sequences were identified. These 63, plus 6 additionalunique fHbp amino acid sequences obtained from the Neisseria.org fHbppeptide database (neisseria.org), were used for the analysis of 69unique fHbp peptides. The respective Genbank accession numbers and/orpeptide identification numbers and the characteristics of the sourcestrains are listed in FIG. 1A-1G (Table 7).

Thirty-eight (55%) of the 69 peptides were classified in the variant 1group of Masignani et al. 2003, 15 (22%) in the variant 2 group, and 16(23%) in the variant 3 group. Of the 69 source strains, one was capsulargroup A, 57 were group B, seven were group C, two were group W-135, andtwo were group X. Multi-locus sequence type (MLST) information wasavailable for 58 of the strains of which 15 were from the ST-269 clonalcomplex, 12 each were from the ST-11, 10 were from the ST-41/44complexes, four were from the ST-162, five were from the ST-213complexes, three each were from the ST-8 and ST-32 complexes. Sixstrains were from other clonal complexes and had sequence types ST-4,ST-35, ST-751, ST-4821, ST-5403 and ST-6874. The 11 strains without MLSTinformation were not available for testing.

In a second study, the dataset further included 172 additionaldistinctive sequences that were subsequently added to the Neisseria.orgdatabase (http://neisseria.org/perl/agdbnet/agdbnet.pl?file=nm_fhbp.xml)as of November 2009. In describing the 242 unique proteins (FIGS.10A-10I, Table 9), the protein identification (ID) numbers from thepeptide database at the Neisseria.org website were employed.

A combination of approaches for analysis of complete or partial proteinsequences was used. Sequences were aligned with MUSCLE (EBI, v3.7,ebi.ac.uk/Tools/muscle/index.html) (Edgar R C. (2004) Nucleic Acids Res.32:1792-7; Edgar R C (2004) BMC Bioinform. 5:113) configured for highestaccuracy. The accuracy of the alignments was confirmed by visualinspection and using the program JALVIEW (Water house A M et al. (2009)Bioinformatics 25:1189-91). Alignments also were performed on theindividual modular variable segments between the blocks of invariantresidues. Networks were generated using SplitsTree, version 4.0 (Huson DH et al., (2006) Mol Biol Evol 23:254-67), with default parameters.Statistical tests for branch support were performed using thebootstrapping method (1000 replicates).

Phylogenic analysis. The analysis of complete or partial proteinsequences was performed on the platform at www.phylogeny.fr (Dereeper etal. Nucleic Acids Res 2008, 36:W465-469) and comprised the followingsteps. Sequences were aligned with MUSCLE (v3.7) (Edgar Nucleic AcidsRes 2004, 32:1792-1797) configured for highest accuracy. The respectivealignments, which contained up to three sites of insertions ordeletions, were inspected and verified by adjacent invariant sequences.The phylogenic tree was reconstructed using the maximum likelihoodmethod implemented in the PhyML program (v3.0 aLRT). Reliability forinternal branch was assessed using the bootstrapping method (100bootstrap replicates). Phylograms were displayed with MEGA 4.0 (Tamuraet al. Mol Biol Evol 2007, 24:1596-1599). The phylograms were rooted onpeptide 1 for consistency. The percent sequence identities within andbetween variable segment types of sequences were determined usingClustalW (Larkin et al. Bioinformatics 2007, 23:2947-2948).

Cloning, expression and purification of recombinant proteins. Expressionplasmids were constructed by PCR amplification of fHbp genes fromgenomic DNA as described previously (Masignani et al. (2003) J Exp Med197:789-99). The genes encoded fHbp ID 1 (Modular group I), 28 (groupII), 22 (group III), 15 (group IV), 79 (group V) and 77 (group VI).C-terminal hexahistidine-tagged recombinant fHbps were expressed inEscherichia coli BL21(DE3) (Novagen, Madison, Wis., US), and purified asdescribed elsewhere (Beernink P T et al. (2008) Infect Immun76:2568-75).

Mouse antisera. Groups of five-week-old CD-1 female mice (10 mice pergroup) were obtained from Charles River (Wilmington, Md., US). The micewere immunized via the intraperitoneal route (IP) with three doses ofvaccine given at 3-week intervals. Each 100-□1 dose contained 20 □g ofrecombinant protein mixed with Freund's adjuvant (FA) (Sigma-Aldrich,St. Louis, Mo., US). (Complete FA for the first dose and incomplete forsubsequent doses). Terminal blood samples were obtained three weeksafter the last dose. The animal procedures were performed under aprotocol approved by the Institutional Animal Care and Use Committee ofthe Children's Hospital Oakland Research Institute.

Bacterial strains. Characteristics of the N. meningitidis strains usedto measure serum bactericidal activity are summarized in Tables 7 and 9.Two isolates each was selected from modular groups Ito VI. For five ofthe modular groups, one strain from each pair was a low expresser offHbp and the other a higher expresser as measured as described below.For modular group V, only strains that expressed intermediate quantitiesof fHbp were identified and, therefore, used for the assays.

Complement-dependent serum bactericidal antibody activity. Thebactericidal assay was performed as described elsewhere, using early logphase bacteria grown for approximately 2 h in Mueller-Hinton broth (BDBiosciences, Franklin Lakes, N.J., US) supplemented with 0.25% glucose(w/v) and 0.02 mM cytidine 5′-monophospho-N-acetylneuraminic acid(Sigma-Aldrich, St, Louis, Mo., US) [11]. The complement source wasserum from a non-immune healthy adult with normal hemolytic complementactivity. The serum was passed over a protein G Sepharose column (HiTrapProtein G HP, GE Healthcare, Piscataway, N.J., US) to remove IgGantibodies (Beernink P T et al. (2009) J Infect Dis 199:1360-8).

Quantitative Western Blotting of fHbp. N. meningitidis cells were grownin broth cultures to early log phase, heat-killed (56° C. for 1 h),collected by centrifugation, and resuspended in PBS to an opticaldensity of 0.6. Proteins in the heat-killed cells (1−4×10⁷ CFU) wereseparated by SDS-PAGE using 4-12% NuPAGE gels (Invitrogen, Carlsbad,Calif., US) as specified by the manufacturer, and were transferred toPVDF membranes (Immobilon-FL; Millipore, Billerica, Mass., US) using aWestern blot module (Invitrogen, Carlsbad Calif., US). After blockingovernight at 4° C. in blocking buffer (Li-Cor Biosciences, Lincoln,Nebr., US), fHbp modular groups I and IV (variant 1 group) were detectedwith anti-fHbp JAR 5 or, JAR 31, for modular groups II, III, V or VI(variant groups 2 or 3) (Beernink PT et la. (2008) Infect Immun76:4243-40). The secondary antibody was goat anti-mouse IgG-IRDye800CW(1:10,000, Rockland Immunochemicals, Gilbertsville, Pa., US).

To determine the quantity of bound protein, the membranes were scannedat 800 nm wavelength using an infrared scanner (Li-Cor Odyssey, Lincoln,Nebr., US), and the integrated intensities of the bands were calculatedwith software provided by the manufacturer (version 3.0.21). The resultsof testing purified, recombinant fHbp from each of the six modulargroups indicated that the IR signal intensities were proportional to thelog₁₀ of the quantities loaded in the range of 0.03 to 2 μg(representative data for control proteins in modular groups I and VI areshown in FIG. 11). Representative expression data are shown for twostrains with fHbp in modular group I, and two strains in modular groupVI (FIG. 11).

Statistical analysis. Statistical calculations were performed usingPrism 5 for Mac OSX, version 5.0a (GraphPad Software, La Jolla, Calif.,US). The proportion of isolates with fHbp in different modular groups ineach country was computed along with the respective 95 percentconfidence intervals calculated from the binomial distribution.Differences found in the proportion of isolates in the respectivemodular groups were compared by Fisher Exact Test (two-tailed) orchi-square analyses.

OVERVIEW OF EXAMPLES

Based on previous analyses of the sequences of the mature peptide, fHbpwas classified into two sub-families (Fletcher et al. Infect Immun 2004,72:2088-2100) or three variant groups (Masignani et al., 2003) (FIG. 3).Analysis presented herein indicated that the overall architecture ofmeningococcal fHbp contains a limited number of specific modularcombinations of five variable segments, with each segment correspondingto one of two types, designated as α or β, based on sequence similarityto the respective segments of fHbp peptides 1 (variant 1 group) or 28(variant 3 group). As described in the Examples section below and shownin FIG. 4, the amino acid sequences of two N. gonorrhoeae orthologs hadarchitectures corresponding to natural chimeras in modular group V (FIG.8). The propensity for Neisseria to transfer genes horizontally (Hotoppet al. Microbiology 2006, 152:3733-3749), coupled with the similarmodular architectures of meningococcal and gonococcal fHbp peptidesdescribed here, suggests that during evolution, the respectiveprogenitor genes recombined at conserved sequences encoding theinvariant blocks of residues hypothesized to be points of junction.

Among the 69 distinct meningococcal fHbp peptides, 65 (94%) could beassigned to one of six modular groups (FIG. 8). Nearly 60% of thepeptides were in modular groups I or II, which comprised only α or βtype segments, respectively. The remaining peptides were naturalchimeras of α and β segments. Modular fHbp groups, III, IV, and V (FIG.8), could each have been generated from the two progenitor types by asingle recombination event. The modular fHbp group VI could have beengenerated by two recombination events. Similarly the four exceptionalsequences described in the Examples section could each have beengenerated through recombination at loci encoding alternative blocks ofother conserved residues. For a protein comprising five segments, eachof which can be of one of two types, there are 2⁵=32 theoreticalindependent modular combinations. That we identified only six modulargroups suggests that there are functional or structural constraints onthe molecule that select for certain combinations of the modules foundin the prevalent fHbp modular groups.

fHbp structural data are available from two recent NMR studies (Cantiniet al., J Biol Chem 2009, doi:10.1074/jbc.C800214200; Mascioni et al. JBiol Chem 2009, doi:10.1074/jbc.M808831200) and a crystallographic studyof fHbp in a complex with a complement regulatory protein (Cantini etal., 2009; Mascioni et al., 2009; Schneider et al. Nature 2009,doi:10.1038/nature07769). In the crystal structure, Schneider et alreported that binding to residues located on the short consensus repeatregion 6 (SCR 6) of fH was mediated by charged amino acid residues inthe fHbp structure that mimicked portions of sugar molecules known tobind to HI (Schneider et al. 2009). Analysis as presented in FIG. 9indicated that these fH contact residues were located on three of thefive variable segments identified in the present study. Of the 21 fHcontact residues, five were invariant among the 69 unique fHbp peptidesanalyzed in our study and ten other contact residues were conserved.

All three fHbp variable segments with fH contact residues also containedamino acids that affected epitopes recognized by bactericidal murineanti-fHbp mAbs (FIG. 9). The central locations of the fH contactresidues in the amino- or carboxyl-terminal domains appeared to bedistinct from the peripheral locations of the residues affecting epitopeexpression. This reciprocal relationship suggests that the fHbpstructures in contact with fH infrequently elicited bactericidalantibodies.

One limitation of fHbp as a vaccine candidate is antigenic variability(Beernink & Granoff, 2009; Masignani et al., 2003). Thus, serumantibodies to recombinant fHbp in the variant 1 group were bactericidalprimarily only against strains with variant 1 proteins (Beernink et al.2007; Fletcher et al. Infect Immun 2004, 72; 2088-2100; Masignani et al.2003), while antibodies to fHbp in the variant 2 or 3 groups hadactivity primarily against strains with homologous variant 2 or 3proteins but had no activity against strains with fHbp in the variant 1group. These observations suggest that epitopes in the C and E segmentsof fHbp, for which variant 1 proteins were phylogenetically separatedfrom those of variant 2 and 3 proteins (FIGS. 6 and 7), are moreimportant for eliciting bactericidal antibodies than those in the A, Bor D segments, where variant 1 proteins clustered together with variant2 and/or 3 proteins (FIGS. 5 and 6). However, all of the variablesegments with the exception of segment B contained residues previouslyidentified as affecting epitopes recognized by murine bactericidalanti-fHbp mAbs (Beernink et al. 2008; Beernink et al. Mol Immunol 2009b,doi:10.1016/j.molimm 2009.02.021; Guiliani et al Infect Immun 2005,73:1151-1160). Further, some of the mAbs inhibited binding of fH to thebacterial surface (Madico et al. J Immunol 2006, 177:501-510), and somecombinations of mAbs that individually were not bactericidal elicitedcooperative bactericidal activity (Beernink et al. 2008; Beernink et al.2009b; Welsch et al. J Infect Dis 2008, 197:1053-1061). The latterincluded mAb JAR 4, which was specific for an epitope on variablesegment A (Beernink et al. 2009b), and elicited cooperative bactericidalactivity with second mAbs with epitopes on variable segment C (forexample JAR 3 or 5) or variable segment E (for example, JAR 13 ormAb502).

To minimize the possibility of fHbp escape mutants, an idealmeningococcal vaccine should include antigenic targets other than fHbp(Giuliani et al., 2006). However, there may be limits to the number ofrecombinant proteins that can be combined in a multicomponent vaccinebecause of the large dose of total protein required, poor tolerability,or loss of immunogenicity of the individual components. To decrease thenumber of individual fHbp components, we engineered chimeric fHbpmolecules containing the amino-terminal domain of a variant 1 fHbp withthe carboxyl-terminal domain of a variant 2 protein using G136 as thejunctional point (Beernink & Granoff, 2008). The resulting chimericprotein expressed epitopes from all three variant groups, and thechimeric fHbp vaccines elicited serum antibodies with bactericidalactivity against a panel of genetically diverse strains expressing fHbpvariant 1, 2 or 3. The junctional point selected for creation of therecombinant chimeric vaccines was located in an invariant portion of themiddle of the variable C segment, which, in the present study, was notidentified as a junctional point for any of the natural fHbp chimericproteins described here. The use of natural junctional points that flankthe C segment may result in improved recombinant chimeric fHbp vaccinesthat elicit broad bactericidal antibodies than other vaccines. It mayalso be possible to introduce amino acid mutations in one or more of thevariable segments to introduce epitopes from heterologous fHbp variants(Beernink & Granoff 2008; Beernink et al. 2009b). Thus, the presence ofsuch fH-binding epitopes in the chimeric fHbp polypeptides can providefor production of antibodies that can facilitate protection against N.meningitidis.

In conclusion, the analysis herein shows that the overall architectureof fHbp can be defined by a limited number of specific modularcombinations of variable segments flanked by invariant sequences.Collectively, the data suggest that recombination occurred between N.meningitidis and N. gonorrhoeae progenitor sequences to generate anantigenically diverse family of meningococcal factor H-binding proteins.The data presented in the Examples thus provide insights into theevolution of fHbp variants, and provide a rational basis forclassification of groups of peptide variants. Population-basedsurveillance studies will be required to define the prevalence of fHbpfrom different modular groups among strains causing carriage and/ordisease, and the relationship between the fHbp modular group and otherstrain characteristics such as capsular group, clonal complex and PorAvariable regions.

Details of the studies that led to this discovery are set out below.

Example 1 Phylogenic Analysis

69 unique fHbp amino acid sequences were aligned to generate a phylogramshown in FIG. 3. For each sequence, the peptide identification numberassigned in fHbp peptide database at neisseria.org is shown and, ifknown, the multi-locus sequence type (MLST) clonal complex is shown inparentheses. The lower left branch shows variant group 1 as defined byMasignani et al 2003 (sub-family B of Fletcher et al 2004); sub-family Acontained two branches, variant groups 2 and 3. Prototype peptides foreach of the variant groups 1, 2 and 3 are indicated with arrows andlabeled with the name of the source strain of the respective genes. Thephylogram was constructed by multiple sequence alignment as described inMethods. The scale bar shown at the bottom indicates 5 amino acidchanges per 100 residues.

The analysis showed the two major branches previously designated assub-families A and B (Fletcher et al. 2004). Sub-family A contained fHbpsequences in antigenic variant groups 2 and 3, and sub-family Bcorresponded to fHbps in the antigenic variant group 1 (Masignani et al.2003). For some clonal complexes, there were examples of strains withfHbp in each of the variant groups (for example, for ST-11, peptides 2,3, 6, 9, 10, 11 and 78 in the variant 1 group, peptides 17, 22, 23 and27 in the variant 2 group and peptide 59 in the variant 3 group). Thestrains from the ST-32 clonal complex had fHbp in two of the variantgroups (peptides 1 and 89 in the variant 1 group and peptide 76 in thevariant 3 group) and the ST-8 clonal complex had fHbp only in onevariant group (peptides 16, 50, and 77 in the variant 2 group). Thedistribution of fHbp variants in all of the observed clonal complexes isgiven in Table 2 below. Peptide IDs listed are assigned by the fHbppeptide database at neisseria.org.

TABLE 2 Distribution of factor H binding protein variants in each clonalcomplex. Clonal Number in Variant group (peptide ID) Complex Variant 1Variant 2 Variant 3 Total ST-4  1 (5) 0 0 1 ST-8  0 3 (16, 50, 77) 0 3ST-11  7 (2, 3, 6, 9, 4 (17, 22, 1 (59) 12 10, 11, 78) 23, 27) ST-32  2(1, 89) 0 1 (76) 3 ST-35  1 (7) 0 0 1 ST-41/44  6 (4, 8, 12, 2 (19, 20)2 (28, 84) 10 14, 86, 87) ST-162  1 (88) 1 (21) 2 (29, 82) 4 ST-213  1(90) 1 (24) 3 (30, 31, 85) 5 ST-269 10 (13, 15, 60-63, 2 (68, 83) 3 (64,70, 72) 15 65, 66, 69, 71) ST-4821  1 (80) 0 0 1 Unassigned  2 (73, 74)0 1 (79) 3 Not done  6 (40, 54-58) 2 (25, 49) 3 (45-47) 11

Example 2 The Architecture of fHbp is Modular

Blocks of two to five invariant residues were identified that separatethe proteins into segments of variable residues. See FIG. 4 for detail.The invariant residues are shown as vertical rectangles. The top threepanels show representative architectures of three N. meningitidis fHbpvariants in groups 1, 2 and 3 (peptide ids, 1, 16 and 28, respectively).Each of the three variants had repetitive amino-terminal elements(N-term), and variable segments A through E, which in some casesdiffered in length among the variants. The amino acid positions of thelast residue in each variable segment are shown.

With the exception of the amino-terminal element, there were identicalrespective invariant amino acid sequences and homologous variablesegments in two N. gonorrhoeae orthologs (Genbank accession numbersAE004969 and CP001050), which overall had 96% amino acid sequenceidentity with meningococcal fHbp peptide 79 (variant 3 meningococcalproteins). In the first study, all 69 fHbp sequence variants had blocksof 2 to 5 invariant residues that flanked five modular variable segments(FIG. 4). These invariant residues were also conserved in 170 of the 172additional fHbp variants analyzed in study 2. One exception, protein ID33, had SHFDF instead of SRFDF between the A and B segments, and theother exception, protein, ID 150, had IEHLE instead of IEHLK between theD and A segments (FIG. 4).

The blocks of invariant residues appeared to represent naturaljunctional points for recombination of the respective genes encoding themeningococcal and gonococcal proteins. The phylogeny of each thevariable segments defined by these junctional points was analyzed. Inthe analysis of the 69 distinctive meningococcal fHbp peptide variantsdescribed below, the numbering of the amino acid residues is based onthe mature fHbp peptide 1 encoded by a gene from MC58 in the variant 1group (Masignani et al. 2003). The numbering for the respective variant2 and 3 proteins differ from that of the variant 1 protein by −1 and +7,respectively (FIG. 4).

Example 3 The Amino-Terminal Repetitive Element

For all 69 sequences, the mature fHbp began with a cysteine residue thatis lipidated by signal peptidase II, which was followed by threeinvariant amino acid residues, SSG. This invariant sequence was followedby a repetitive variable sequence consisting of 1 to 6 glycine and/orserine residues and then by two invariant glycine residues (FIG. 4). Thevariable portion of the amino-terminal element consisted of a singleglycine residue for 34 of the 69 peptides (nearly all in the variant 1group, Table 3 shown below), or GG residues for 12 of the peptides (allin the variant 2 group). The other common variable sequence was GGGSGG(8 in the variant 1 group and 7 in the variant 3 group).

TABLE 3 Sequence and distribution of amino-terminal repetitive elementVariable Number of Peptides in Variant Group Sequence¹ Variant 1Variant 2 Variant 3 G 29 3 2 GG 0 12 0 SGG 0 0 1 GGGS 0 0 2(SEQ ID NO: 7) SGSGG 0 0 1 (SEQ ID NO: 8) GGGSGG 8 0 6 (SEQ ID NO: 9)GGGSGS 1 0 4 (SEQ ID NO: 10) ¹The mature fHbp begins with an invarianttetrapeptide, CSSG, which is followed by a repetitive variable sequenceconsisting of 1 to 6 glycine and/or serine residues, which is thenfollowed by two invariant glycine residues.

Example 4 Downstream Variable Segments

There were five variable segments, which we designated A, B, C, D or E(FIG. 4). Based on the presence of certain signature amino acid residuesand sequence similarity (Table 4), each of the five segments could besegregated into one of two types. One of the types had signature aminoacid residues and sequence similarity to peptides in the antigenicvariant 1 group. The other type had signature amino acid residues andsequence similarity to peptides in the antigenic variant 3 group, whichalso were similar to those of the gonococcal ortholog (see FIG. 4). Alsosee Table 4 presented below.

TABLE 4 Amino acid identity within and between sequence groups bysegment. α Sequence Types β Sequence Types Identity Identity Identitybetween α Variable Invariant Signature Range Signature Range and β TypesSegment Residue Range¹ Block² residues³ No.⁴ (%) residues³ No.⁴ (%)⁴(%)⁴ A  8-73 SRFDF QSV 48 89-100 DSI and 21 89-100⁵ 69-78⁵ KDN B 79-93GEFQ IRQ 53 80-100⁵ VQK 16 100 60-66⁵ C  98-159 DD QDS 38 85-100 NNP 3193-100 32-45 D 162-180 IEHLK AG or 58 89-100 PN 11 84-100 52-63 AS E186-253 KQ⁶ SLGI 38 86-100 HLAL 31 94-100⁵ 48-57⁵ ¹Amino acid numberingbased on the mature fHbp from strain MC58. ²Invariant block of residuesimmediately following each variable segment. The invariant sequenceimmediately before segment A was GG (See FIG. 4). ³See FIG. 2A-2E:(Table 8) for complete sequences of each variable segment. ⁴Number ofsequences in each sequence type for each variable segment (N = 61peptides). ⁵Four fHbp sequences with exceptional junctional positions atother conserved blocks of residues were excluded from the percentidentity analyses (see text). ⁶Corresponds to carboxyl-terminal end offHbp sequence

For purposes of classification, the first group of segments isdesignated as α types and the second group as β types. The amino acididentity of the respective segments within an α or β type ranges from 80to 100% (Table 4). In contrast, the identity of segments between typesranged from 69-78% for segment A, which was located in a conservedregion of the N-terminal portion of the molecule, to 32-45% in the Csegment, which encompassed resides 98-159 in a much less conservedregion of the protein (Table 4). For each segment, distinct sequencevariants were assigned a unique identifier beginning with a letter, Athrough E, to represent the variable segment; followed by an α or β toindicate the presence of residues with the respective types describedabove, followed by a number for each distinct sequence (listed in FIG.2A-2E (Table 8)).

Segment A began at amino acid residue 8 immediately after the invariantGG sequence and extended to position 73 (Table 4). Among the 69 fHbpvariants, segment A contained 16 distinctive a sequence variants, and 9distinctive β sequence variants (FIG. 5). Where multiple peptidespossessed an identical sequence in a segment, the number of peptides isgiven in parentheses. The scale bar indicates 5 amino acid changes per100 residues. Based on the phylogram, the A segments of most of thefHbps in the variant 1 group (shown in FIG. 5) clustered with those inthe variant 2 group, whereas the A segments of fHbps in the variant 3group were in a separate cluster. The most common α variant (N=14) wasdesignated A.α.1, which was present in 4 fHbp peptides in the variant 1group and 10 in the variant 2 group. The most common β variant wasdesignated A.β.1, which was present in 6 peptides, all in the variant 3group.

Segment B began at position 79 immediately after an invariant SRFDFsequence and extended to position 93 (Table 4). Among the 69 fHbppeptides, segment B contained 7 different α variant sequences and asingle β sequence (FIG. 5). The phylogenic analysis showed that the Bsegments of fHbp peptides in the variant 1 and 2 groups clusteredtogether (FIG. 5), which were distinct from those in the variant 3group. The most common B segment, B.α.1, was present in 40 fHbp peptides(28 in the variant 1 group and 12 in the variant 2 group).

In contrast to the A and B segments, the respective C (residues 98-159)and E (residues 186-253) segments of fHbp in the variant 2 and 3 groupsclustered together and were separated from those of fHbps in the variant1 group 1 (FIG. 6). All of the D segments (residues 162-180) of strainswith fHbp in the variant group 1 were a types while those of fHbppeptides in the variant 2 or 3 groups were α or β (FIG. 7). The mostcommon D segment, D.α.1 (N=39), was present in 21 fHbp v.1 peptides, 9v.2 peptides and 9 v.3 peptides.

Example 5 Classification of fHbp Variants by Modular Groups

Based on the phylogenic analysis of the variable segments of fHbpdescribed above, the majority of these different fHbp variants studiedwere categorized into six distinct fHbp modular groups (I to VI) (FIG.8). Forty of the 69 fHbp peptides (58%) comprised only a (N=33) or β(N=7) type segments, which were designated fHbp modular groups I and II,respectively (FIG. 8). The α segments are shown in gray and the βsegments are shown in white. The remaining 29 peptides (42%) could beclassified into one of four chimeras derived from recombination ofdifferent α or β segments (designated fHbp modular groups III, IV, V orVI; N=25) or, as described below, were other chimeras (N=4) in which oneof the five segments used an exceptional junctional position at otherconserved blocks of sequences. As seen in FIG. 6, a representativepeptide in each modular group is indicated by the peptide identificationnumber from the fHbp database at neisseria.org. The number of uniquesequences observed within each fHbp modular group is indicated at theright. This analysis excluded four peptides sequences with exceptionaljunctional points at one of their segments.

Although the amino-terminal repetitive segment described above was notused to define the modular fHbp groups, the majority (88 to 100%) ofmodular group I, III and VI peptides sequences had amino-terminalrepetitive elements of 1 or 2 glycine residues (Table 3). In contrast,the majority (75 to 100%) of the peptides in modular groups II, IV and Vhad amino-terminal elements of 3 to 6 glycine and serine residues. Thelength of this amino-terminal element may affect the distance of theprotein from the bacterial membrane and surface-accessibility of certainepitopes.

In the second study, all but three of the 172 new fHbp variants could beclassified into one of the six modular groups (I to VI, FIG. 4, PanelA). The three exceptions (protein ID 207, 67 and 175) had distinctivecombinations of modular segments derived from α or β type lineages andwere assigned to one of three new modular groups (VII, VIII and IX,respectively).

Of 242 distinct fHbp variants in the expanded database, 125 proteinswere in modular group I (FIG. 4), which contained only α segments, and20 proteins were in modular group II (entirely (segments). The remaining97 fHbp sequence variants were natural chimeras of α and β segments andwere assigned to modular groups III through IX (together, 40 percent ofall variants). FIG. 4 shows the fHbp variant groups as described byMasignani et al. (2003) J Exp Med 1997:789-99 for each of the modulargroups. The variant groups were assigned based on the relatedness of theoverall amino acid sequences of the respective proteins.

Example 6 Chimeric fHbp Peptides Containing Junction Points Within aVariable Segment

Four fHbp sequences had modular structures similar to those describedabove except that one of the junctional points between two of thesegments utilized alternative invariant sequences. For example, fHbppeptide 55 was similar to peptides in modular group IV (FIG. 8) exceptthat the A segment switched from a β type sequence to an α type sequenceat an invariant AQGAE starting at residue 50 rather than at SRFDFstarting at residue 74. This A segment was designated A.β.9 (β becauseof its higher sequence identity to other β type A segments than to the αtype A segments; FIG. 5). Two other fHbp peptides, 24 and 25 (FIG. 2A-2E(Table 8)), had modular structures similar to peptides in modular groupIII (FIG. 8) but had B segments that switched from an α type to a β typesequence at an invariant IEV beginning at residue 82 instead of GEFQ atposition 94. This exceptional B segment, designated B.α.3, wascategorized as an α type because of its higher sequence identity toother a type B segments than to the β type B segments (FIG. 5). Thefourth exceptional fHbp modular structure, peptide 82 (FIG. 2A-2E (Table8)), was similar to type V, except that its E segment, designatedE.β.10, switched from an α type sequence to a β type at residue A196instead of at IEHLK starting at position 181 (FIG. 6).

Example 7 Structural Features of the Variable and Invariant fHbpSegments

The respective variable and invariant segments of fHbp were mapped ontoa molecular model based on the published coordinates from the fHbpcrystal structure (FIG. 9) (Schneider et al. 2009). The amino- andcarboxyl-termini, labeled N and C, respectively, in panel A are inidentical positions in panels B and C. The figure was constructed withPyMol (pymol.org). The models in the center of panels A, B and C havebeen rotated 180 degrees on the Y-axis from the corresponding models onthe far left, while the models on the far right have been rotated 90degrees on the X-axis as compared with the models in the middle.

The ribbon models in panel A show the two previously described majordomains of fHbp (Cantini et al. 2009; Mascioni et al. 2009; Schneider etal. 2009), each containing independently folded beta-structures, whichare connected together by a structured, four amino acid residue linkerlocated in variable segment C. The amino-terminal domain, which beginsat residue 8 and extends to residue 136, includes variable segments Aand B and part of C (note that the first 14 residues of the matureprotein are not present in the crystal structure). The carboxyl-terminaldomain, which extends from residue 141 to 255, includes thecarboxyl-terminal portion of the C and the entire D and E variablesegments.

Panel B shows the corresponding space-filled models. The amino acidresidues previously reported to be in contact with fH based on thestructure of the fHbp-fH complex are depicted in black. These residuesform clusters on variable segments A, C and E and are visible in themodels shown in the middle and far right of panel B. Since fH is knownto bind to fHbp on live bacteria (Madico et al. 2006; Schneider et la.2006), this binding site must be surface-exposed. The invariant blocksof residues that flank each of the five variable segments, are localizedon the opposite face of the protein and shown in white in panels B and C(far left model). Since the amino-terminus, which contains the signalanchor, extends from this face of the protein (bottom surface of modelat far right, panel B), the invariant residues are predicted to belocated entirely on the surface of the molecule anchored to the cellwall. The presence of these invariant sequences on themembrane-associated surface suggests that there are structuralconstraints, perhaps a requirement for a partner protein, for anchoringand/or orienting fHbp on the bacterial cell membrane.

Previous studies mapped the epitopes of eleven bactericidal anti-fHbpmAbs (Beernink et al. 2008; Beernink et al. 2009b; Giuliani et al. 2005;Scarselli et al J Mol Biol 2009, 386:97-108). In panel C (FIG. 9), theamino acids affecting expression of each of these epitopes are depictedin black and the previously defined fH contact residues (shown in blackas in panel B). With the exception of segment B, all of the variablesegments contained epitopes recognized by bactericidal mAbs. The aminoacids affecting the epitopes generally were located on the periphery ofthe amino- and carboxyl-terminal domains of fHbp whereas the residues incontact with fH were located in clusters in the central portions of eachof the two domains. The epitopes of certain mAbs such as JAR 3, 5 or 13,which were previously reported to inhibit binding of fH to fHbp(Beernink et al. 2008; Beernink et al. 2009b), involved amino acidslocated in proximity to some of the fH contact residues. However, therewas no example of overlap between the two sets of residues.

Example 8 Frequency of fHbp Modular Groups Among Isolates CausingDisease in Different Countries

The analysis described above provided information on the extent of fHbpmodular group diversity. For this following analysis, the fHbp sequencedata from systematically collected group β isolates in the U.S. andEurope reported by Murphy et al. (2009) J Infect Dis 200:379-89 wereused. The isolates were from cases in the United States between 2001 and2005 (N=432), and from the United Kingdom (N=536), France (N=244),Norway (N=23) and the Czech Republic (N=27) for the years 2001 to 2006.The U.S. data were supplemented with fHbp sequences of 143 additionalisolates that had been systematically collected at multiple sites in theU.S. as part of another study (Beernink P T et al. (2007) J Infect Dis195:1472-9).

Among the total of 1405 systematically collected group β isolates,modular group I was found in 59.7%, group II in 1.7%, group III in 8.1%,group IV in 10.6%, group V in 6.1%, and group VI in 13.6%. The newmodular groups, VII, VIII and IX, were each found in ≦0.1%. Therespective distributions of the fHbp modular groups in the differentcountries as stratified by the variant group classification of Masignaniet al. (2003) J Exp Med 197:789-99 are shown in FIG. 12. For thisanalysis, the data from Norway and Czech Republic were excluded, sincethe number of isolates in each of these collections was too small forprecise estimates of the respective frequencies of the modular groups.The respective percentages of the modular groups in the two U.S.collections are shown separately.

Isolates in variant 1 group of Masignani consisted of modular groups I,IV and VII. Modular group I strains, which have entirely a-typesegments, predominated in all three countries (54 to 64 percent of allisolates). In the UK, however, 23% of all isolates were modular groupIV, which are natural chimeras of α- and β-type segments (FIG. 8), ascompared with <1% in the two U.S. collections, and 3 percent of isolatesfrom France (P<0.001 by chi square). Since there was only one isolate inmodular group VII (France), the frequency of this modular group is notshown in FIG. 12.

Isolates in variant 2 group included modular groups III and VI, whichare all natural chimeras of α- and β-type segments. Modular groups IIIor VI were present in approximately equal proportions of isolates in thetwo U.S. collections, while modular group VI predominated among variant2 isolates from the UK and France.

Isolates with variant 3 group included modular group II (entirely β-typesegments) and modular groups V, VIII and IX, which are chimeras. InFrance and the UK, modular group V accounted for the majority of theisolates with variant 3 fHbp, while in the two U.S. collections therewere approximately equal numbers of modular group II or V proteins.Modular groups VIII and IX accounted for <0.1% of the isolates. Becauseof the low percentages, these groups are not shown on FIG. 12.

Example 9 Strain Susceptibility to Anti-fHbp Serum Bactericidal Activityin Relation to Modular Group and fHbp Expression

FIG. 13 depicts human complement bactericidal titers of serum pools frommice immunized with recombinant fHbp vaccines representative of modulargroups I-VI. The heights of the bars represent the respective mediantiters of each of the six antisera (3 to 4 pools per modular group) whentested against the specific test strain. For example, the upper leftpanel shows the data for strain H44/76, which is a high expresser offHbp in modular group I (relative expression is designated by +++;actual values are shown in Table 5 below). The median bactericidal titerof the homologous anti-fHbp modular group I antiserum (black bar) was˜1:6000. The respective titers of the five heterologous anti-fHbpmodular group antisera (white bars) against strain H44/76 were 1 to 2log_(in) lower, ranging from <1:10 (antisera to modular group II) to˜1:200 (antisera to modular group IV). The corresponding median titersof the anti-modular group II and IV antisera when tested against controlstrains with homologous fHbp modular groups II (strain SK104 in theVariant 3 panel) or IV (NM452 in the Variant 1 panel) were >1:2000.Thus, these and the other heterologous antisera had high antibodyactivity when measured against control strains with the respectivehomologous fHbp modular groups.

There were lower bactericidal titers against low fHbp expressing strainsthan against high expressing strains (compare respective graphs on theright side of FIG. 13 showing data for low expressing strains[designated +/−], with those on the left showing higher expressers). Forexample the anti-modular group I antiserum had a titer of 1:6000 againstH44/76, a high fHbp modular group I expressing strain, but a titer of˜1:100 against strain 03S-0408, a low fHbp modular group I expresser.(For modular group V, both test strains were intermediate expressers,since a pair of high and low fHbp expressing strains were notavailable).

There was a trend for lower cross-reactivity of anti-fHbp bactericidalactivity against strains with low expression of fHbp from heterologousmodular groups than for high expressing strains (see for example, datafor low expressing strains 03S-0408 (modular group I), M01573 (modulargroup IV), and RM1090 (modular group III), which were killed only bytheir respective homologous modular group antisera as compared with thebroader bactericidal activity against the respective higher fHbpexpressing strains (H44/76, NM452, and 03S-0673; FIG. 13). Strain NMB(fHbp modular group VI) was completely resistant to anti-fHbpbactericidal activity (titer <1:10), which apparently was a result oflow fHbp expression (Table 5). The amino acid sequence of the vaccineused to prepare the anti-modular group VI antisera differed by a singleamino acid from fHbp expressed by NMB, and the resulting antisera werebactericidal against a higher expressing strain (961-5945) with fHbpmodular group VI. Further, the resistant strain, NMB, was killed by acontrol anti-PorA mAb at a concentration of 0.15 μg/ml, which wasidentical to that of an anti-PorA mAb required for killing of theanti-fHbp susceptible 961-5945 strain.

TABLE 5 Characteristics of N. meningitidis strains used for measuringserum bactericidal activity. Factor H-binding protein Expression GenbankStrain sequence clonal Variant Protein Modular (ng/10⁶ accession nametypes complex group ID group cells) number H44/76 32 32 1 1 I 31AY548370 03S-0408 11 11 1 78 I 3 ACF35432 SK104 5748 162 3 99 II 11GQ219769 M1239 437 41/44 3 28 II 2 ABF82029 03S-0673 1364 32 2 23 III 14GU056306 RM1090 11 11 2 22 III 2 ABY26518 NM452 283 269 1 15 IV 20ABL14232 M01573 44 41/44 1 55 IV 1 AAR84481 S3032 6874 n/a 3 79 V 8ACH48234 MD1475 162 162 3 82 V 7 GQ219772 961-5945 153 8 2 16 VI 5AAR84453 NMB 8 8 2 50 VI 1 AY330379

The antibodies used for the quantitative measurement of fHbp and thereactivity of various modular group proteins for each antibody is shownin the Table below.

TABLE 6 Anti-fHbp monoclonal antibody reactivity of recombinant proteinsused to prepare antisera in relationship to locations of epitopes.Anti-fHbp mAb JAR 1 JAR 4 JAR 3 or JAR 10 JAR 11 JAR 13 JAR 31 JAR 32JAR 33 JAR 5 JAR 35 Epitope Amino R204 (25-27) G121, K180 A174 S216 Unk⁶K174 R180 location acid YGN K122 and and residue (57-59) E192⁴ E192⁴ (s)KDN Modular Eα Aα Cα Dα and Dα Eβ UD⁶ Dβ Dβ and segment Eβ Eβ ModularVariant Protein Group Group ID No. Reactivity of Recombinant Protein byELISA⁵ I 1 1 1 1 1 0 0 0 0 0 0 II 3 28 0 0 0 0 0 1 1 1 1 III 2 22 0 1 00 0 0 1 1 1 IV 1 15 0 0 1 0 0 0 0 0 0 V 3 79 0 0 0 1 1 1 1 0 0 VI 2 77 01 0 1 1 1 1 0 0 ¹All of the MAbs were bactericidal when tested incertain combinations with human complement ³Discontinuous epitope;presence of KDN suppresses epitope [1] ⁴Discontinuous epitope requiringspecific combinations of charged residues ⁵Assigned “1” if Opticaldensity >3 SD above background binding when tested at 1 μg/ml, “0”,negative. ⁶UD, location of epitope undertermined

1. A non-naturally occurring fHbp, comprising, from N-terminus toC-terminus:V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E), wherein the combination ofalleles for each of V_(A), V_(B), V_(C), V_(D), and V_(E) variablesegments is not found in nature.
 2. The non-naturally occurring fHbp ofclaim 1, wherein each of said variable segments is an α progenitorsequence.
 3. The non-naturally occurring fHbp of claim 1, wherein eachof said variable segments is a β progenitor sequence.
 4. Thenon-naturally occurring fHbp of claim 1, wherein at least one of V_(A),V_(B), V_(C), V_(D), and V_(E) is of an α progenitor fHbp amino acidsequence and at least one of V_(A), V_(B), V_(C), V_(D), and V_(E) is ofan β progenitor fHbp amino acid sequence.
 5. The non-naturally occurringfHbp of claim 4, wherein said non-naturally occurring fHbp is not of amodular group Type as set forth in FIG.
 8. 6. The non-naturallyoccurring fHbp of claim 1, where said non-naturally occurring fHbpcomprises, from N-terminus to C-terminus:I₁-Nte-I₂-V_(A)-I₃-V_(B)-I₄-V_(C)-I₅-V_(D)-I₆-V_(E)-I₇.
 7. Thenon-naturally occurring fHbp of claim 1, where said non-naturallyoccurring fHbp comprises, from N-terminus to C-terminus:V_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)β-I₆-V_(E)α orV_(A)α-I₃-V_(B)α-I₄-V_(C)α-I₅-V_(D)β-I₆-V_(E)β.
 8. The non-naturallyoccurring fHbp of claim 1, wherein said fHbp comprises an epitope thatis bound by a monoclonal antibody set forth in Table
 1. 9. A method ofeliciting an antibody response in a mammal, the method comprisingadministering to a mammal a composition comprising a first non-naturallyoccurring fHbp according to claim
 1. 10. The method of claim 9, whereinthe mammal is a human.
 11. The method of claim 9, wherein saidadministering provides for production of antibodies that bind to two ormore types of fHbp.
 12. The method of claim 9, wherein saidnon-naturally occurring fHbp is in a preparation comprising outermembrane vesicles (OMVs), microvesicles (MVs), or a mixture of OMVs andMVs.
 13. The method of claim 12, wherein said vesicles are prepared fromtwo or more strains of N. meningitidis, each of which is geneticallydifferent from the other.
 14. The method of claim 9, wherein saidadministering comprises administering a second fHbp, wherein said secondfHbp is different from said first non-naturally occurring fHbp.
 15. Animmunogenic composition comprising a first non-naturally occurring fHbpaccording to claim 1, and a pharmaceutically acceptable excipient. 16.The immunogenic composition of claim 15, further comprising a secondfHbp, wherein said second fHbp is different from said firstnon-naturally occurring fHbp.
 17. The immunogenic composition of claim15, wherein said non-naturally occurring fHbp is in a vesiclepreparation, wherein vesicles are prepared from a Neisseria bacterium.18. The immunogenic composition of claim 17, wherein said Neisseriabacterium is genetically modified to disrupt production of an endogenousfHbp polypeptide.
 19. The immunogenic composition of claim 15, whereinsaid non-naturally occurring fHbp is produced by a Neisseria bacteriumthat is genetically modified to produce said fHbp.
 20. The immunogeniccomposition of claim 18, wherein said vesicle preparation is from thesame Neisseria meningitidis strain as the strain producing saidnon-naturally occurring fHbp.
 21. The immunogenic composition of claim18, wherein said Neisseria bacterium is deficient in capsularpolysaccharide synthesis.
 22. A nucleic acid encoding the non-naturallyoccurring fHbp of claim
 1. 23. A host cell containing the nucleic acidof claim 22.